Her-2 targeted bispecific compositions and methods for making and using the same

ABSTRACT

Disclosed herein are polypeptides, and methods of making and using such polypeptides, that comprise a bispecific antibody construct covalently linked to an extended recombinant polypeptide comprising a barcode fragment releasable from said polypeptide upon digestion by a protease, and a Release Segment that can be proteolytically cleaved wherein said cleavage releases the bispecific antibody construct from the extended recombinant polypeptide.

RELATED APPLICATIONS

This application is a continuation of International Patent ApplicationNo. PCT/US2021/039006, filed Jun. 24, 2021, which claims priority toU.S. Provisional Patent Application Ser. Nos. 63/044,301, filed Jun. 25,2020; 63/077,503, filed Sep. 11, 2020; 63/108,783, filed Nov. 2, 2020;63/166,857, filed Mar. 26, 2021; and 63/196,408, filed Jun. 3, 2021, theentire disclosures of which are hereby incorporated herein by reference.

SEQUENCE LISTING STATEMENT

The instant application contains a Sequence Listing which has beensubmitted electronically in XML format and is hereby incorporated byreference in its entirety. Said XML file, created on Dec. 10, 2022, isnamed 735659_SA9-739PCCON_ST26.xml and is 2,734,656 bytes in size.

BACKGROUND

Bispecific T Cell Engagers (TCEs) present a highly potent modality forcancer therapy that redirects T-cell cytotoxicity against tumors thatexpress a selected tumor-associated antigen, bypassing the requirementsfor T-cell recognition of tumor antigens. The activity of a TCE dependson its ability to activate T cells through effective stimulation of theT cell receptor (TCR). Their extreme potency derives from the minimalrequirement for as few as 3 TCRs to become stimulated and coalesce toform an immune synapse between the T cell and target cell to initiatecytotoxicity. In addition to their induction of cytotoxicity, theirpotency also involves cytokine-driven actions downstream of T-cellactivation that enhance and amplify the anti-tumor immune response.Thus, TCEs offer the promise of immunotherapy to patients whose tumorsharbor insufficient mutations or have escaped immune surveillance byother means. However, this modality is not without its own challengesand the use of TCEs in solid tumors has been limited by their extremepotency and on target, off-tumor toxicities in healthy tissue.

TCEs have been highly effective at inducing remission in patients withhematologic cancers, but their use in solid tumors has been limited bytheir extreme potency and on-target toxicities against normal tissueexpressing even low levels of target. Impressive, but rare clinicalresponses have been observed against tumors typically refractory toimmunotherapy (e.g., microsatellite-stable colorectal and prostatecancers), but toxicities such as cytokine release syndrome at low doseshave prevented dose escalation to reveal the modality's clinicalpotential. Grade 4 cytokine release syndrome induced in patients treatedwith the Ichnos ISB 1302 HER2 TCE at <1 ug/kg doses highlights thechallenge faced by TCEs even when directed against relativelytissue-restricted targets.

Clinical trials with blinatumomab, (an approved CD3×CD19 bispecificantibody) revealed that cytokine release syndrome (CRS) is one of themajor safety-related adverse events. CRS and on-target toxicities at lowdrug doses have significantly compromised the therapeutic index andpotential of the TCE modality in the clinic against solid tumors. Forexample, the clinical trial for catumaxomab (CD3×EpCAM) was terminateddue to drug-related hepatic failure at a 10 μg dose. In another trial, aHER2-targeted TCE (Glenmark GBR1302) the dose of the agent was limitedto less than 1 μg/kg due to onset of G4 CRS. While pasotuxumab (aPSMA-targeted TCE) showed a good response, it was hampered by CRS atdoses greater than 40 μg/day. The literature is replete with otherexamples of the CRS and on-target toxicity challenges presented by TCEs.

Attempts to circumvent CRS include complex molecular designs, but thesehave been unsuccessful due to toxicity and/or enhanced immunogenicity.This presents a significant unmet need for new strategies that canovercome therapeutic index challenges in solid tumors. If the potency ofTCEs could be harnessed and the CRS and on-target toxicity challengescould be controlled, it may be possible to generate powerfultherapeutics that could potentially be used against a broad spectrum ofcancers.

A therapeutic, such as a drug substance includes polypeptides that maybe produced in a manner that results in a mixture of polypeptides thatcan influence activity of the drug substance. The mixture ofpolypeptides can often include the full-length polypeptide, along withsize variants (e.g., truncations) thereof. The presence of variants thatdiffer in size from the desired full-length product may affect thebiological behavior of a drug substance, potentially affecting thesafety and/or efficacy of the polypeptide drug substance. For example,protein-based prodrugs for cancer therapy may be engineered with atumor-targeted activation mechanism. More specifically, the full-lengththerapeutic protein may be an inactive (non-cytotoxic) prodrug form,while truncation variants of the full-length construct may loseprotective sequences and become cytotoxic (active), thus “contaminating”the prodrug composition. In some instances, such shorter length variantsmay pose a greater risk of immunogenicity, have less selective toxicityfor tumor cells, or show a less desired pharmacokinetic profile (e.g.,resulting in a narrowed therapeutic window) compared with thefull-length protein. As a result, detection and quantification ofprotein structural variations can be of importance in assessingbiological properties (e.g., clinical safety and pharmacologic efficacy)of biotherapeutics and in developing new biotherapeutics (e.g., withincreased efficacy and reduced side effects). Existing techniques andmethods for identifying and quantifying the amount of “contaminating”truncation products may include one or more drawbacks, such as being oflimited sensitivity, ease, efficiency, or effectiveness.

SUMMARY

The present invention addresses a long-felt unmet need in providing TCEcancer therapeutics that have an increased therapeutic index. In doingso, the invention harnesses the therapeutic potential of TCEs byproviding XTENylated protease activated bispecific T cell engagers(XPATs). XPATs represent a novel strategy to improve the toxicityprofile of T cell engagers while maintaining their potency against solidtumors, thus enabling a significant increase in the therapeutic indexand expansion of target landscape for this potent modality. In certainspecific embodiments, the XPATs of the present invention targetHER2-bearing tumors. More specifically, AMX-818 is a HER2-targeted,conditionally activated prodrug TCE designed to exploit the dysregulatedprotease activity in tumors, while sparing healthy tissues whereprotease inhibition prevails, thus broadening the safety margin andtherapeutic index

Provided herein is a polypeptide having an N-terminal amino acid and aC-terminal amino acid, the polypeptide comprising: (a) an extendedrecombinant polypeptide (XTEN), comprising a barcode fragment (BAR)releasable from said polypeptide upon digestion by a protease; (b) abispecific antibody construct (BsAb), comprising a first antigen bindingfragment (AF1) that specifically binds to a cluster of differentiation 3T cell receptor (CD3), which AF1 comprises light chaincomplementarity-determining regions 1 (CDR-L1), 2 (CDR-L2), and 3(CDR-L3) and heavy chain complementarity-determining regions 1 (CDR-H1),2 (CDR-H2), and 3 (CDR-H3), wherein said CDR-H3 comprises an amino acidsequence of SEQ ID NO:10; and a second antigen binding fragment (AF2)that specifically binds to human epidermal growth factor receptor 2(HER2); and (c) a release segment (RS) positioned between said XTEN andsaid bispecific antibody construct, wherein said XTEN is characterizedin that: (i) it comprises at least 100, or at least 150 amino acids;(ii) at least 90% of its amino acid residues are glycine (G), alanine(A), serine (S), threonine (T), glutamate (E) or proline (P); (iii) itcomprises at least 4 different types of amino acids that are G, A, S, T,E, or P; and (iv) said XTEN is formed from a plurality ofnon-overlapping sequence motifs that are each from 9 to 14 amino acidsin length, wherein said plurality of non-overlapping sequence motifscomprise: (1) a set of non-overlapping sequence motifs, wherein eachnon-overlapping sequence motif of said set of non-overlapping sequencemotifs is repeated at least two times in said XTEN; and (2) anon-overlapping sequence motif that occurs only once within said XTEN;wherein said barcode fragment (BAR) includes at least part of saidnon-overlapping sequence motif that occurs only once within said XTEN;wherein said barcode fragment (BAR) differs in sequence and molecularweight from all other peptides fragments that are releasable from saidpolypeptide upon complete digestion of said polypeptide by saidprotease; and wherein said barcode fragment (BAR) does not include saidN-terminal amino acid or said C-terminal amino acid of said polypeptide.

In certain embodiments, said set of non-overlapping sequence motifs eachindependently comprise an amino acid sequence identified herein by SEQID NOS: 179-200 and 1715-1722. In certain embodiments, said set ofnon-overlapping sequence motifs each independently comprise an aminoacid sequence identified herein by SEQ ID NOS: 186-189. In certainembodiments, said set of non-overlapping sequence motifs comprise atleast two, at least three, or all four of the sequence motifs SEQ IDNOS: 186-189. In certain embodiments, said XTEN comprises a length offrom 100 to 3,000, from 150 to 3,000, from 100 to 1,000, or from 150 to1,000 amino acid residues. In certain embodiments, said XTEN comprises alength of at least 200, at least 250, at least 300, at least 350, atleast 400, at least 450, or at least 500 amino acid residues. In certainembodiments, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or100% of the amino acid residues of said XTEN are glycine (G), alanine(A), serine (S), threonine (T), glutamate (E) or proline (P).

In certain embodiments, said XTEN has at least 90%, at least 92%, atleast 95%, at least 98%, at least 99% or 100% sequence identity to asequence set forth in Table 3a. In certain embodiments, said barcodefragment (BAR) does not include a glutamic acid that is immediatelyadjacent to another glutamic acid, if present, in said XTEN. In certainembodiments, said barcode fragment (BAR) has a glutamic acid at itsC-terminus. In certain embodiments, said barcode fragment (BAR) has anN-terminal amino acid that is immediately preceded by a glutamic acidresidue. In certain embodiments, said barcode fragment (BAR) ispositioned a distance from either said N-terminus of said polypeptide orsaid C-terminus of said polypeptide, wherein said distance is from 10 to150 amino acids, or from 10 to 125 amino acids in length. In certainembodiments, said barcode fragment (BAR) is characterized in that: (i)it does not include a glutamic acid that is immediately adjacent toanother glutamic acid, if present, in said XTEN; (ii) it has a glutamicacid at its C-terminus; (iii) it has an N-terminal amino acid that isimmediately preceded by a glutamic acid residue; and (iv) it ispositioned a distance from either said N-terminus of said polypeptide orsaid C-terminus of said polypeptide, wherein said distance is from 10 to150 amino acids, or from 10 to 125 amino acids in length. In certainembodiments, said glutamic acid residue that precedes said N-terminalamino acid of said barcode fragment (BAR) is not immediately adjacent toanother glutamic acid residue.

In certain embodiments, said barcode fragment (BAR) does not include asecond glutamic acid residue at a position other than the C-terminus ofsaid barcode fragment unless said second glutamic acid is immediatelyfollowed by a proline. In certain embodiments, said XTEN is positionedN-terminal of said bispecific antibody construct (BsAb), wherein saidbarcode fragment (BAR) is positioned within 200, within 150, within 100,or within 50 amino acids of said N-terminus of said polypeptide. Incertain embodiments, said XTEN is positioned N-terminal of saidbispecific antibody construct (BsAb), and wherein said barcode fragment(BAR1) is positioned at a location that is between 10 and 200, between30 and 200, between 40 and 150, or between 50 and 100 amino acids fromsaid N-terminus of said protein. In certain embodiments, said XTEN ispositioned C-terminal of said bispecific antibody construct (BsAb), andwherein said barcode fragment (BAR) is positioned within 200, within150, within 100, or within 50 amino acids of said C-terminus of saidpolypeptide. In certain embodiments, said XTEN is positioned C-terminalof said bispecific antibody construct (BsAb), and said barcode fragment(BAR) is positioned at a location that is between 10 and 200, between 30and 200, between 40 and 150, or between 50 and 100 amino acids from saidC-terminus of said protein. In certain embodiments, said barcodefragment (BAR) is at least 4 amino acids in length. In certainembodiments, said barcode fragment (BAR) is between 4 and 20, between 5and 15, between 6 and 12, or between 7 and 10 amino acids in length.

In certain embodiments, said barcode fragment (BAR) comprises an aminoacid sequence set forth in Table 2. In certain embodiments, said XTENhas a length defined by a proximal end and a distal end, wherein (1)said proximal end is positioned, relative to said distal end, closer tosaid bispecific antibody construct (BsAb), and wherein (2) said barcodefragment (BAR) is positioned within a region of said XTEN that extends,as measured from said distal end, between 5% and 50%, between 7% and40%, or between 10% and 30% of said length of said XTEN. In certainembodiments, said XTEN further comprises additional one or more barcodefragments, wherein said additional one or more barcode fragments eachdiffer in sequence and molecular weight from all other peptidesfragments that are releasable from said polypeptide upon completedigestion of said polypeptide by said protease. In certain embodiments,said release segment (RS) comprises an amino acid sequence having atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequenceidentity to a sequence identified herein by SEQ ID NOS: 7001-7626. Incertain embodiments, said protease cleaves on the C-terminal side ofglutamic acid residues that are not followed by proline. In certainembodiments, said protease is a Glu-C protease.

In certain embodiments, the polypeptide is expressed as a fusionprotein, wherein said fusion protein, in an uncleaved state, has astructural arrangement from N-terminus to C-terminus that isAF1-AF2-RS-XTEN, AF2-AF1-RS-XTEN, XTEN-RS-AF1-AF2, or XTEN-RS-AF2-AF1.In certain embodiments, said release segment (RS) is fused to saidbispecific antibody construct (BsAb) by a spacer. In certainembodiments, said spacer comprises at least 4 types of amino acids thatcan be glycine (G), alanine (A), serine (S), threonine (T), glutamate(E) or proline (P). In certain embodiments, said spacer comprises anamino acid sequence having at least 80%, 90%, or 100% sequence identityto a sequence set forth in Table C. In certain embodiments, said CDR-H1and said CDR-H2 of said first antigen binding fragment (AF1) compriseamino acid sequences of SEQ ID NOS: 8 and 9, respectively.

In certain embodiments, said CDR-L1 of said AF1 comprises an amino acidsequence of SEQ ID NO:1 or 2; said CDR-L2 of said AF1 comprises an aminoacid sequence of SEQ ID NO:4 or 5; and said CDR-L3 of said AF1 comprisesan amino acid sequence of SEQ ID NO:6. In certain embodiments, saidCDR-L1 of said AF1 comprises an amino acid sequence of SEQ ID NO:1; saidCDR-L2 of said AF1 comprises an amino acid sequence of SEQ ID NO:4 or 5;and said CDR-L3 of said AF1 comprises an amino acid sequence of SEQ IDNO:6. In certain embodiments, said CDR-L1 of said AF1 comprises an aminoacid sequence of SEQ ID NO:2; said CDR-L2 of said AF1 comprises an aminoacid sequence of SEQ ID NO:4 or 5; and said CDR-L3 of said AF1 comprisesan amino acid sequence of SEQ ID NO:6. In certain embodiments, saidCDR-L1 of said AF1 comprises an amino acid sequence of SEQ ID NO:1; saidCDR-L2 of said AF1 comprises an amino acid sequence of SEQ ID NO:4; andsaid CDR-L3 of said AF1 comprises an amino acid sequence of SEQ ID NO:6.In certain embodiments, said CDR-L1 of said AF1 comprises an amino acidsequence of SEQ ID NO:2; said CDR-L2 of said AF1 comprises an amino acidsequence of SEQ ID NO:5; and said CDR-L3 of said AF1 comprises an aminoacid sequence of SEQ ID NO:6. In certain embodiments, said first antigenbinding fragment (AF1) comprises four chain variable domain frameworkregions 1 (FR-H1), 2 (FR-H2), 3 (FR-H3), and 4 (FR-H4), each exhibitingat least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% sequence identity or being identical to an amino acidsequence of SEQ ID NOS: 60, 64, 65, and 67, respectively. In certainembodiments, said first antigen binding fragment (AF1) comprises fourchain variable domain framework regions 1 (FR-H1), 2 (FR-H2), 3 (FR-H3),and 4 (FR-H4), each exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or beingidentical to an amino acid sequence of SEQ ID NOS: 61, 64, 65, and 67,respectively. In certain embodiments, said first antigen bindingfragment further comprises four light chain variable domain frameworkregions (FR-L): FR-L1, FR-L2, FR-L3, and FR-L4, each exhibiting at least86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity or being identical to amino acid sequences of SEQ IDNOS: 51, 52, 53, and 59, respectively. In certain embodiments, saidfirst antigen binding fragment further comprises four light chainvariable domain framework regions (FR-L): FR-L1, FR-L2, FR-L3, andFR-L4, each exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% sequence identity or being identical toamino acid sequences of SEQ ID NOS: 51, 52, 54, and 59, respectively.

In certain embodiments, said first antigen binding fragment furthercomprises four light chain variable domain framework regions (FR-L):FR-L1, FR-L2, FR-L3, and FR-L4, each exhibiting at least 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequenceidentity or being identical to amino acid sequences of SEQ ID NOS: 51,52, 55, and 59, respectively. In certain embodiments, said first antigenbinding fragment further comprises four light chain variable domainframework regions (FR-L): FR-L1, FR-L2, FR-L3, and FR-L4, eachexhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% sequence identity or being identical to amino acidsequences of SEQ ID NOS: 51, 52, 56, and 59, respectively. In certainembodiments, said first antigen binding fragment (AF1) further compriseslight chain framework regions 1 (FR-L1), 2 (FR-L2), 3 (FR-L3), and 4(FR-L4) and heavy chain framework regions 1 (FR-H1), 2 (FR-H2), 3(FR-H3) and 4 (FR-H4), wherein: said FR-L1 comprises an amino acidsequence of SEQ ID NO:51; said FR-L2 comprises an amino acid sequence ofSEQ ID NO:52; said FR-L3 comprises an amino acid sequence of SEQ IDNO:53, 54, 55, or 56; said FR-L4 comprises an amino acid sequence of SEQID NO:59; said FR-H1 comprises an amino acid sequence of SEQ ID NO:60 or61; said FR-H2 comprises an amino acid sequence of SEQ ID NO:64; saidFR-H3 comprises an amino acid sequence of SEQ ID NO:65; and said FR-H4comprises an amino acid sequence of SEQ ID NO:67.

In certain embodiments, said first antigen binding fragment (AF1)further comprises light chain framework regions 1 (FR-L1), 2 (FR-L2), 3(FR-L3), and 4 (FR-L4) and heavy chain framework regions 1 (FR-H1), 2(FR-H2), 3 (FR-H3) and 4 (FR-H4), wherein: said FR-L1 comprises an aminoacid sequence of SEQ ID NO:51; said FR-L2 comprises an amino acidsequence of SEQ ID NO:52; said FR-L3 comprises an amino acid sequence ofSEQ ID NO:53; said FR-L4 comprises an amino acid sequence of SEQ IDNO:59; said FR-H1 comprises an amino acid sequence of SEQ ID NO:60; saidFR-H2 comprises an amino acid sequence of SEQ ID NO:64; said FR-H3comprises an amino acid sequence of SEQ ID NO:65; and said FR-H4comprises an amino acid sequence of SEQ ID NO:67.

In certain embodiments, said first antigen binding fragment (AF1)further comprises light chain framework regions 1 (FR-L1), 2 (FR-L2), 3(FR-L3), and 4 (FR-L4) and heavy chain framework regions 1 (FR-H1), 2(FR-H2), 3 (FR-H3) and 4 (FR-H4), wherein: said FR-L1 comprises an aminoacid sequence of SEQ ID NO:51; said FR-L2 comprises an amino acidsequence of SEQ ID NO:52; said FR-L3 comprises an amino acid sequence ofSEQ ID NO:54; said FR-L4 comprises an amino acid sequence of SEQ IDNO:59; said FR-H1 comprises an amino acid sequence of SEQ ID NO:61; saidFR-H2 comprises an amino acid sequence of SEQ ID NO:64; said FR-H3comprises an amino acid sequence of SEQ ID NO:65; and said FR-H4comprises an amino acid sequence of SEQ ID NO:67.

In certain embodiments, said first antigen binding fragment (AF1)further comprises light chain framework regions 1 (FR-L1), 2 (FR-L2), 3(FR-L3), and 4 (FR-L4) and heavy chain framework regions 1 (FR-H1), 2(FR-H2), 3 (FR-H3) and 4 (FR-H4), wherein: said FR-L1 comprises an aminoacid sequence of SEQ ID NO:51; said FR-L2 comprises an amino acidsequence of SEQ ID NO:52; said FR-L3 comprises an amino acid sequence ofSEQ ID NO:55; said FR-L4 comprises an amino acid sequence of SEQ IDNO:59; said FR-H1 comprises an amino acid sequence of SEQ ID NO:61; saidFR-H2 comprises an amino acid sequence of SEQ ID NO:64; said FR-H3comprises an amino acid sequence of SEQ ID NO:65; and said FR-H4comprises an amino acid sequence of SEQ ID NO:67.

In certain embodiments, said first antigen binding fragment (AF1)further comprises light chain framework regions 1 (FR-L1), 2 (FR-L2), 3(FR-L3), and 4 (FR-L4) and heavy chain framework regions 1 (FR-H1), 2(FR-H2), 3 (FR-H3) and 4 (FR-H4), wherein: said FR-L1 comprises an aminoacid sequence of SEQ ID NO:51; said FR-L2 comprises an amino acidsequence of SEQ ID NO:52; said FR-L3 comprises an amino acid sequence ofSEQ ID NO:56; said FR-L4 comprises an amino acid sequence of SEQ IDNO:59; said FR-H1 comprises an amino acid sequence of SEQ ID NO:61; saidFR-H2 comprises an amino acid sequence of SEQ ID NO:64; said FR-H3comprises an amino acid sequence of SEQ ID NO:65; and said FR-H4comprises an amino acid sequence of SEQ ID NO:67.

In certain embodiments, said first antigen binding fragment (AF1)exhibits a higher thermal stability than an anti-CD3 binding fragmenthaving a sequence set forth in SEQ ID NO: 206, as evidenced in an invitro assay by a higher melting temperature (T_(m)) of said firstantigen binding fragment relative to that of said anti-CD3 bindingfragment; or upon incorporating said first antigen binding fragment intoa test bispecific antigen binding construct, a higher T_(m) of said testbispecific antigen binding construct relative to that of a controlbispecific antigen binding construct, wherein said test bispecificantigen binding construct comprises said first antigen binding fragmentand a reference antigen binding fragment that binds to an antigen otherthan CD3; and wherein said control bispecific antigen binding constructconsists of said anti-CD3 binding fragment consisting of said sequenceof SEQ ID NO:206 and said reference antigen binding fragment. In certainembodiments, said T_(m) of said first antigen binding fragment is atleast 2° C. greater, or at least 3° C. greater, or at least 4° C.greater, or at least 5° C. greater than said T_(m) of said anti-CD3binding fragment consisting of said sequence of SEQ ID NO:206. Incertain embodiments, said first antigen binding fragment (AF1) comprisesa heavy chain variable region (VH_(I)), wherein said VH_(I) comprises anamino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% sequence identity or being identical to an amino acidsequence of SEQ ID NO:102 or 105. In certain embodiments, said firstantigen binding fragment (AF1) comprises a light chain variable region(VL_(I)), wherein said VL_(I) comprises an amino acid sequence having atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identityor is identical to an amino acid sequence of any one of SEQ ID NOS: 101,103, 104, 106, or 107. In certain embodiments, said VH_(I) and saidVL_(I) is linked by a linker comprising an amino acid sequence having atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequenceidentity to a sequence set forth in Table A. In certain embodiments,said first antigen binding fragment (AF1) comprises an amino acidsequence having at least 95%, 96%, 97%, 98%, 99% sequence identity orbeing identical to an amino acid sequence of any one of SEQ ID NOS:201-205. In certain embodiments, said first antigen binding fragment(AF1) specifically binds human or cynomolgus monkey (cyno) CD3. Incertain embodiments, said first antigen binding fragment (AF1)specifically binds human CD3. In certain embodiments, said first antigenbinding fragment (AF1) binds a CD3 complex subunit that is CD3 epsilon,CD3 delta, CD3 gamma, or CD3 zeta unit of CD3. In certain embodiments,said first antigen binding fragment (AF1) binds a CD3 epsilon fragmentof CD3. In certain embodiments, said first antigen binding fragment(AF1) exhibits an isoelectric point (pI) that is less than or equal to6.6. In certain embodiments, said first antigen binding fragment (AF1)exhibits an isoelectric point (pI) that is between 6.0 and 6.6,inclusive.

In certain embodiments, said first antigen binding fragment (AF1)exhibits an isoelectric point (pI) that is at least 0.1, 0.2, 0.3, 0.4,0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 pH units lower than the pI of areference antigen binding fragment having a sequence shown in SEQ ID NO:206. In certain embodiments, said first antigen binding fragment (AF1)specifically binds human or cyno CD3 with a dissociation constant(K_(d)) constant between about between about 10 nM and about 400 nM, asdetermined in an in vitro antigen-binding assay comprising a human orcyno CD3 antigen. In certain embodiments, said first antigen bindingfragment (AF1) specifically binds human or cyno CD3 with a dissociationconstant (K_(d)) of less than about 10 nM, or less than about 50 nM, orless than about 100 nM, or less than about 150 nM, or less than about200 nM, or less than about 250 nM, or less than about 300 nM, or lessthan about 350 nM, or less than about 400 nM, as determined in an invitro antigen-binding assay. In certain embodiments, said first antigenbinding fragment (AF1) exhibits a binding affinity to CD3 that is atleast 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, orat least 10-fold weaker relative to that of an antigen binding fragmentconsisting of an amino acid sequence of SEQ ID NO:206, as determined bythe respective dissociation constants (K_(d)) in an in vitroantigen-binding assay.

In certain embodiments, said first antigen binding fragment (AF1) is achimeric or a humanized antigen binding fragment. In certainembodiments, said first antigen binding fragment (AF1) is Fv, Fab, Fab′,Fab′-SH, linear antibody, or single-chain variable fragment (scFv). Incertain embodiments, said second antigen binding fragment (AF2) is Fv,Fab, Fab′, Fab′-SH, linear antibody, a single domain antibody, orsingle-chain variable fragment (scFv). In certain embodiments, saidfirst and second antigen binding fragments are configured as an (Fab′)₂or a single chain diabody. In certain embodiments, said second antigenbinding fragment (AF2) comprises a heavy chain variable region (VH_(II))comprising an amino acid sequence identified herein by SEQ ID NOS:778-783, and a light chain variable region (VL_(II)) comprising an aminoacid sequence identified herein by SEQ ID NOS: 878-883. In certainembodiments, said VH_(II) and said VL_(II) is linked by a linkercomprising an amino acid sequence having at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence setforth in Table A. In certain embodiments, said first and second antigenbinding fragments are fused together by a peptide linker. In certainembodiments, said peptide linker comprises 2 or 3 types of amino acidsthat are glycine, serine, or proline. In certain embodiments, saidpeptide linker comprises an amino acid sequence having at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity toa sequence set forth in Table B.

In certain embodiments, said XTEN is a first extended recombinantpolypeptide (XTEN1); formed from a plurality of non-overlapping sequencemotifs, comprising a first plurality of non-overlapping sequence motifs;said BAR is a first barcode fragment (BAR1); and said RS is a firstrelease segment (RS1); and the polypeptide further comprising: (d) asecond extended recombinant polypeptide (XTEN2), comprising a secondbarcode fragment (BAR2) releasable from said polypeptide upon digestionby said protease; and (e) a second release segment (RS2) positionedbetween said second XTEN (XTEN2) and said bispecific antibody construct(BsAb), wherein said XTEN2 is characterized in that: (i) it comprises atleast 100, or at least 150 amino acids; (ii) at least 90% of its aminoacid residues that are glycine (G), alanine (A), serine (S), threonine(T), glutamate (E) or proline (P); and (iii) it comprises at least 4different types of amino acids that are G, A, S, T, E, or P; whereinsaid second barcode fragment (BAR2) differs in sequence and molecularweight from all other peptides fragments that are releasable from saidpolypeptide upon complete digestion of said polypeptide by saidprotease; and wherein said second barcode fragment (BAR2) does notinclude said N-terminal amino acid or said C-terminal amino acid of saidpolypeptide. In certain embodiments, said XTEN1 is positioned N-terminalof said bispecific antibody construct and said XTEN2 is positionedC-terminal of said bispecific antibody construct. In certainembodiments, said XTEN1 is positioned C-terminal of said bispecificantibody construct and said XTEN2 is positioned N-terminal of saidbispecific antibody construct.

In certain embodiments, said XTEN2 is formed from a second plurality ofnon-overlapping sequence motifs that are each from 9 to 14 amino acidsin length, wherein said second plurality of non-overlapping sequencemotifs comprise: (1) a second set of non-overlapping sequence motifsrepeated at least two times in said second XTEN; and (2) anon-overlapping sequence motif that occurs only once within said secondXTEN; and wherein said second barcode fragment (BAR2) includes at leastpart of said non-overlapping sequence motif that occurs only once withinsaid second XTEN. In certain embodiments, said second set ofnon-overlapping sequence motifs are each independently identified hereinby SEQ ID NOS: 179-200 and 1715-1722. In certain embodiments, saidsecond set of non-overlapping sequence motifs are each independentlyidentified herein by SEQ ID NOS: 186-189. In certain embodiments, saidsecond set of non-overlapping sequence motifs comprise at least two, atleast three, or all four of the sequence motifs SEQ ID NOS: 186-189. Incertain embodiments, said XTEN2 comprises a length of from 100 to 3,000,from 150 to 3,000, from 100 to 1,000, or from 150 to 1,000 amino acidresidues. In certain embodiments, said XTEN2 comprises a length of atleast 200, at least 250, at least 300, at least 350, at least 400, atleast 450, or at least 500 amino acid residues.

In certain embodiments, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or 100% of the amino acid residues of said XTEN2 are glycine (G),alanine (A), serine (S), threonine (T), glutamate (E) or proline (P). Incertain embodiments, said XTEN2 has at least 90%, at least 92%, at least95%, at least 98%, at least 99% or 100% sequence identity to a sequenceset forth in Table 3a. In certain embodiments, said second barcodefragment (BAR2) does not include a glutamic acid that is immediatelyadjacent to another glutamic acid, if present, in said XTEN2. In certainembodiments, said second barcode fragment (BAR2) has a glutamic acid atits C-terminus. In certain embodiments, said second barcode fragment(BAR2) has an N-terminal amino acid that is immediately preceded by aglutamic acid residue. In certain embodiments, said second barcodefragment (BAR2) is positioned a distance from either said N-terminus ofsaid polypeptide or said C-terminus of said polypeptide, wherein saiddistance is from 10 to 150 amino acids, or from 10 to 125 amino acids inlength. In certain embodiments, said second barcode fragment (BAR2) ischaracterized in that (i) it does not include a glutamic acid that isimmediately adjacent to another glutamic acid, if present, in saidXTEN2; (ii) it has a glutamic acid at its C-terminus; (iii) it has anN-terminal amino acid that is immediately preceded by a glutamic acidresidue; and (iv) it is positioned a distance from either saidN-terminus of said polypeptide or said C-terminus of said polypeptide,wherein said distance is from 10 to 150 amino acids, or from 10 to 125amino acids in length.

In certain embodiments, said glutamic acid residue that precedes saidN-terminal amino acid of said BAR2 is not immediately adjacent toanother glutamic acid residue. In certain embodiments, said secondbarcode fragment (BAR2) does not include a second glutamic acid residueat a position other than the C-terminus of said second barcode fragment(BAR2) unless said second glutamic acid is immediately followed by aproline. In certain embodiments, said XTEN2 is positioned N-terminal ofsaid bispecific antibody construct (BsAb), and wherein said secondbarcode fragment (BAR2) is positioned within 200, within 150, within100, or within 50 amino acids of said N-terminus of said polypeptide. Incertain embodiments, said XTEN2 is positioned N-terminal of saidbispecific antibody construct (BsAb), and wherein said second barcodefragment (BAR2) is positioned at a location that is between 10 and 200,between 30 and 200, between 40 and 150, or between 50 and 100 aminoacids from said N-terminus of said protein. In certain embodiments, saidXTEN2 is positioned C-terminal of said bispecific antibody construct(BsAb), and wherein said second barcode fragment (BAR2) is positionedwithin 200, within 150, within 100, or within 50 amino acids of saidC-terminus of said polypeptide. In certain embodiments, said XTEN2 ispositioned C-terminal of said bispecific antibody construct (BsAb), andsaid second barcode fragment (BAR2) is positioned at a location that isbetween 10 and 200, between 30 and 200, between 40 and 150, or between50 and 100 amino acids from said C-terminus of said protein.

In certain embodiments, said second barcode fragment (BAR2) is at least4 amino acids in length. In certain embodiments, said second barcodefragment (BAR2) is between 4 and 20, between 5 and 15, between 6 and 12,or between 7 and 10 amino acids in length. In certain embodiments, saidsecond barcode fragment (BAR2) comprises an amino acid sequence as setforth in Table 2. In certain embodiments, said XTEN2 has a lengthdefined by a proximal end and a distal end, wherein (1) said proximalend of said XTEN2 is positioned, relative to said distal end, closer tosaid bispecific antibody construct (BsAb), and wherein (2) said secondbarcode fragment (BAR2) is positioned within a region of said XTEN2 thatextends, as measured from said distal end of said XTEN2, between 5% and50%, between 7% and 40%, or between 10% and 30% of said length of saidXTEN2. In certain embodiments, said XTEN2 further comprises additionalone or more barcode fragments, wherein said additional one or morebarcode fragments of said XTEN2 each differ in sequence and molecularweight from all other peptides fragments that are releasable from saidpolypeptide upon complete digestion of said polypeptide by saidprotease. In certain embodiments, said first release segment (RS1) andsaid second release segment (RS2) are identical in sequence. In certainembodiments, said first release segment (RS1) and said second releasesegment (RS2) are not identical in sequence. In certain embodiments,said second release segment (RS2) comprises an amino acid sequencehaving at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%sequence identity to a sequence identified herein by SEQ IDNOS:7001-7626. In certain embodiments, said first release segment (RS1)and said second release segment (RS2) are each a substrate for cleavageby multiple proteases at one, two, or three cleavage sites within eachrelease segment sequence.

In certain embodiments, a polypeptide is expressed as a fusion protein,wherein said fusion protein, in an uncleaved state, has a structuralarrangement from N-terminus to C-terminus identified herein byXTEN1-RS1-AF1-AF2-RS2-XTEN2, XTEN1-RS1-AF2-AF1-RS2-XTEN2,XTEN2-RS2-AF1-AF2-RS1-XTEN1, XTEN2-RS2-AF2-AF1-RS1-XTEN1,XTEN1-RS1-diabody-RS2-XTEN2, or XTEN2-RS2-diabody-RS1-XTEN1, whereinsaid diabody comprises a light chain variable region (VL_(I)) of saidAF1, a heavy chain variable region (VH_(I)) of said AF1, a light chainvariable region (VL_(II)) of said AF2, and a heavy chain variable region(VH_(II)) of said AF2. In certain embodiments, said spacer of said firstrelease segment (RS1) is a first spacer, and wherein said second releasesegment (RS2) is fused to said bispecific antibody construct (BsAb) by asecond spacer. In certain embodiments, said second spacer comprises atleast 4 types of amino acids that are glycine (G), alanine (A), serine(S), threonine (T), glutamate (E) or proline (P). In certainembodiments, said second spacer comprises an amino acid sequence havingat least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%sequence identity to a sequence set forth in Table C.

In certain embodiments, said XTEN1 comprises an amino acid sequencehaving at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100% sequence identity to a sequence set forth in Table 3a; said BsAbcomprises: said AF1 comprising light chain complementarity-determiningregions 1 (CDR-L1), 2 (CDR-L2), and 3 (CDR-L3) and heavy chaincomplementarity-determining regions 1 (CDR-H1), 2 (CDR-H2), and 3(CDR-H3), wherein said CDR-H1, said CDR-H2, and said CDR-H3 compriseamino acid sequences of SEQ ID NOS: 8, 9, and 10, respectively; and saidAF2 comprising a light chain variable region (VL_(II)) identified hereinby SEQ ID NOS: 778-783 and a heavy chain variable region (VH_(II))identified herein by SEQ ID NOS: 878-883; said RS1 comprises an aminoacid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% sequence identity to a sequence identified herein bySEQ ID NOS: 7001-7626; said XTEN2 comprises an amino acid sequencehaving at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100% sequence identity to a sequence set forth in Table 3a; and saidRS2 comprises an amino acid sequence having at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequenceidentified herein by SEQ ID NOS: 7001-7626, wherein said polypeptide hasa structural arrangement from N-terminus to C-terminus identified hereinas XTEN1-RS1-AF2-AF1-RS2-XTEN2, XTEN1-RS1-AF1-AF2-RS2-XTEN2,XTEN2-RS2-AF2-AF1-RS1-XTEN1, or XTEN2-RS2-AF1-AF2-RS1-XTEN1.

In certain embodiments, the polypeptide has a terminal half-life that isat least two-fold longer compared to the bispecific antibody constructnot linked to any XTEN. In certain embodiments, the polypeptide is lessimmunogenic compared to the bispecific antibody construct not linked toany XTEN, as ascertained by measuring production of IgG antibodies thatselectively bind to said bispecific antibody construct afteradministration of comparable doses to a subject. In certain embodiments,the polypeptide exhibits an apparent molecular weight factor underphysiological conditions that is greater than about 3, greater thanabout 4, greater than about 5, or greater than about 6. In certainembodiments, the polypeptide comprises an amino acid sequence having atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequenceidentity to a sequence identified herein as the sequences in Table D.

In certain embodiments, a pharmaceutical composition comprises apolypeptide as above and one or more pharmaceutically suitableexcipients. In certain embodiments, said pharmaceutical composition isformulated for administration to a human or animal by any clinicallyappropriate route and formulation. In certain embodiments, saidpharmaceutical composition is in a liquid form or frozen. In certainembodiments, said pharmaceutical composition is in a pre-filled syringefor a single injection. In certain embodiments, said pharmaceuticalcomposition is formulated as a lyophilized powder to be reconstitutedprior to administration.

In certain embodiments said composition is in a pharmaceuticalcombination with at least one additional therapeutic agent selected fromthe group consisting of an antibody, an antibody fragment, an antibodyconjugate, a cytotoxic agent, a toxin, a radionuclide, animmunomodulator, a photoactive therapeutic agent, a radiosensitizingagent, a hormone, an anti-angiogenesis agent, and combinations thereof.

The additional therapeutic agent may be a PD-1/PD-L1(2) inhibitorwherein the PD-1/PD-L1(2) inhibitor is an anti-PD-1 antibody or ananti-PD-L1 antibody or an anti-PD-L2 antibody.

In certain embodiments the PD-1/PD-L1(2) inhibitor is an anti-PD-1antibody selected from the group comprising nivolumab (Opdivo,BMS-936558, MDX1106), pembrolizumab (Keytruda, MK-3475, lambrolizumab),pidilizumab (CT-011), PDR-001, JS001, STI-A1110, AMP-224 and AMP-514(MEDI0680).

In one embodiment the PD-1/PD-L1(2) inhibitor can be an anti-PD-L1antibody selected from the group comprising atezolizumab (Tecentriq,MPDL3280A), durvalumab (MEDI4736), avelumab (MSB0010718C), BMS-936559(MDX1105) and LY3300054. In another embodiment the PD-1/PD-L1(2)inhibitor is an anti-PD-L2 antibody.

In certain embodiments the combination is a combination pack containingthe components separate from one another. In certain embodiments thecomponents are administered in separate dosage forms simultaneously orsequentially for use in the treatment of the same disease.

In certain embodiments the polypeptide described herein is part of apharmaceutical combination for use as medicament for treatinghyper-proliferative disorders. The hyper-proliferative disorders areselected from the group consisting of cancers of the breast, respiratorytract, brain, reproductive organs, digestive tract, urinary tract, eye,liver, skin, head and neck, thyroid, parathyroid and their distantmetastases.

In certain embodiments, a polypeptide as described herein is used in thepreparation of a medicament for the treatment of a disease in a subject.In certain embodiments, said disease is cancer.

In certain embodiments is a method of treating a disease in a subject,comprising administering to said subject in need thereof one or moretherapeutically effective doses of a pharmaceutical composition or apharmaceutical combination.

In one embodiment the disease is cancer. In certain embodiments thecancer is selected from the group consisting of glioblastoma, melanoma,cholangio carcinoma, small cell lung cancer, colorectal cancer, prostatecancer, vaginal cancer, angiosarcoma, non-small cell lung cancer,appendiceal cancer, squamous cell cancer, salivary duct carcinoma,adenoid cystic carcinoma, small intestine cancer, and gallbladdercancer.

Provided herein is a method of treating a disease in a subject,comprising administering to said subject in need thereof one or moretherapeutically effective doses of a pharmaceutical composition of asabove. In certain embodiments, the pharmaceutical composition isadministered to the subject as one or more therapeutically effectivedoses administered during a therapeutically effective course oftreatment. In certain embodiments, the dose is administered to a humanor animal by any clinically appropriate route and formulation. Incertain embodiments, the subject is a mouse, rat, monkey, or human.

Also provided herein is a nucleic acid comprising a polynucleotidesequence encoding a polypeptide as described herein; or a reversecomplement of said polynucleotide sequence thereof. Further providedherein is an expression vector comprising said polynucleotide sequenceof as described herein and a recombinant regulatory sequence operablylinked to said polynucleotide sequence. Provided herein is a host cell,comprising said expression vector. In certain embodiments, the host cellis a prokaryote. In certain embodiments, the host cell is E. coli. Incertain embodiments, the host cell is a mammalian cell.

Additional aspects and advantages of the present disclosure will becomereadily apparent to those skilled in this art from the followingdetailed description, wherein only illustrative embodiments of thepresent disclosure are shown and described. As will be realized, thepresent disclosure is capable of other and different embodiments, andits several details are capable of modifications in various obviousrespects, all without departing from the disclosure. Accordingly, thedrawings and description are to be regarded as illustrative in nature,and not as restrictive.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

Various features of this disclosure are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present disclosure will be obtained by reference tothe following detailed description that sets forth illustrativeembodiments, in which the principles of the invention are utilized, andthe accompanying drawings of which:

FIG. 1 depicts a mixture of XTENylated Protease-Activated T Cell Engager(“XPAT”) polypeptides having varying lengths of XTEN. The full-lengthXPAT (top) comprises a 288 amino acid-long XTEN at the N-terminus and a864 amino acid-long XTEN at the C-terminus. Various truncations canoccur in the XPAT in one or both of the N- and C-terminal XTENs, forexample, during fermentation, purification or other steps in productpreparation. While products having limited truncations (truncations neara distal end an XTEN) may function in a manner similar to thefull-length construct, severe truncations (truncations closer to theproximal end of the XTEN) may possess significantly differentpharmacological properties from their full-length counterparts. Thepresence of truncations poses a challenge for quantifying thepharmacologically efficacious and inefficacious variants in an XPATproduct. As illustrated in FIG. 1 using the full-length XPAT, each XTENhas a proximal end and a distal end, wherein the proximal end ispositioned, relative to the distal end, closer to the biologicallyactive polypeptide (e.g., T-cell engager, cytokine, monoclonal antibody(mAb), antibody fragment, or other protein that is XTENylated).

FIG. 2 depicts a mixture of XPAT polypeptides having varying lengths ofbarcoded XTEN. In the full-length XPAT (top), the 288 amino acid-longN-terminal XTEN contains three cleavably fused barcode sequences, “NA,”“NB,” and “NC” (from distal end to proximal end), and the 864 aminoacid-long C-terminal XTEN contains three cleavably fused barcodesequences, “CC,” “CB,” and “CA” (from proximal end to distal end). Eachbarcode is positioned to indicate a pharmacologically-relevant length ofthe corresponding XTEN. For example, minor N-terminal truncationproducts of the XPAT, lacking the barcode “NA” (e.g., due to truncation)but having the more proximal barcodes “NB” and “NC,” may showsubstantially the same pharmacological properties as the full-lengthconstruct. In contrast, major N-terminal truncation products of theXPAT, e.g., lacking all three barcodes (e.g., due to truncation) on theN-terminus, may discernibly differ in pharmacological activity from thefull-length construct. A unique proteolytically-cleavable sequence isidentified from the biologically-active polypeptide (here, the tandemscFvs that comprise the active portion of the T-cell engager) of theXPAT. Due to its presence in all the length variants of the XPAT(including full-length XPAT, minor truncations, and major truncationsthereof), the unique proteolytically-cleavable sequence can be used as areference for quantifying the amounts of various truncation products inrelation to the total amount of the biologically active protein.

FIG. 3 illustrates a potential design for a barcoded XTEN by inserting abarcode-generating sequence into a general-purpose (or regular) XTEN.The exemplary general-purpose (or regular) XTEN (top) comprisesnon-overlapping 12-mer motifs in the sequence “BCDABDCDABDCBDCDABDCB,”wherein the sequence motifs “A,” “B,” “C,” and “D” occur 3, 6, 5, and 7times, respectively. Glu-C protease digest of the exemplarygeneral-purpose XTEN (upper panel) does not yield unique peptides exceptboth termini (“NT” and “CT”). The insertion of a barcode-generatingsequence, “X” (e.g., a unique 12-mer), into the XTEN results in a uniqueproteolytically-cleavable sequence (or barcode sequence) that does notoccur anywhere else in the XTEN. The barcode-generating sequence, “X,”can be positioned such that the resulting barcode marks apharmacologically-relevant length of the XTEN. For example, an XTENlacking a barcode due to truncation may functionally differ from thecorresponding XTEN with the barcode. One of ordinary skill in the artwill understand that the barcode-generating sequence (“X”) can be thebarcode sequence itself. Alternatively, the barcode-generating sequence(“X”) can differ from the resulting barcode sequence. For example, thebarcode sequence can overlap with and, thus, contain part of thepreceding or following 12-mer motif.

FIGS. 4A-4B illustrate the quantification of the level of truncation foran N-terminal XTEN. FIG. 4A demonstrates that a barcoded XTEN (bottompanel) can be constructed by replacing a sequence motif in ageneral-purpose XTEN (top panel) (e.g., the third sequence motif fromthe N-terminus, “D”) with a barcode-generating motif, “X”; and, in thisexample, the barcode-generating motif (“X”) is itself the uniqueproteolytically-cleavable barcode sequence. As shown in the bottom panelin FIG. 4A, the barcode is positioned such that all the severetruncation forms of the XTEN lack the barcode, and all the limitedtruncation forms of the XTEN contain the barcode. FIG. 4B illustratesthe relative abundance of various cleavage products in two differentmixtures of XPAT. In one of the mixtures, the barcode is present in 99%of the constructs that contain the biologically active protein. In theother one of the mixtures, 13% of the constructs are lack a barcode(e.g., due to truncation). FIGS. 4A-4B illustrate the use of barcodedXTEN to differentiate between two polypeptide mixtures havingsubstantially similar average molecular weights but discerniblydifferent pharmacological activities.

FIGS. 5A-5B and 6A-6C illustrate dose-dependent cytotoxicity of masked(XTENylated) and unmasked bispecific T-cell engagers against targetcells with various levels of target antigen expression.Proteolytically-unmasked (de-XTENylated) bispecifics demonstrate potentcytotoxicity against various tumor lines, for example, with EC50s in thesingle-digit pM range when tested with SK-OV-3 and BT-474 cells.XTENylation further demonstrate robust masking capability, e.g., inshielding a bispecific T-cell engager from forming an immune synapse,thereby resulting in reduced toxicity as indicated by a right-ward shiftin the concentration-response curve.

FIGS. 5A-5B illustrate effective masking by XTENs on XTENylatedProtease-Activated T-cell engagers (“XPATs”) in general, and onHER2-XPATs in particular. For example, cytotoxicity of the XTENylated(masked) HER2-XPAT (e.g., set forth in Table D) and the correspondingde-XTENylated (unmasked, activated) HER2-PAT against two differentHER2-expressing (e.g., cancer) cell lines was observed in adose-dependent manner. The unmasked, de-XTENylated PAT (indicated assolid circles) yielded EC50 values of 3.4 picomolar (pM) (SKOV3 cells)(FIG. 5A) and 4.8 pM (BT474 cells) (FIG. 5B) respectively, while thecorresponding masked, XTENylated PAT (indicated as solid squares)yielded EC50 values of 44,474 pM (SKOV3 cells) and 49,370 pM (BT474cells), indicating a masking effect of at least 10⁴-fold.

FIG. 6A depicts effective masking by XTENs on a bispecific T-cellengager when in contact with non-cancerous tissue (cardiomyocytes). Moreparticularly, FIG. 6A shows the cytotoxicity of the XTENylated (masked)HER2-XPAT (e.g., set forth in Table D) and the correspondingde-XTENylated (unmasked, activated) HER2-PAT against cardiomyocytes.Killing of the cardiomyocytes by T cell-directed cytolysis in responseto unmasked, de-XTENylated PAT was observed (with an approximate EC50concentration of 64 pM), while in contrast to tumor cells, thecardiomyocytes remained refractory to killing by masked, XTENylated PATat concentrations as high as 1 micromolar (μM).

FIGS. 6B-6C illustrate robust masking by XTENs on bispecific T-cellengagers in the context of engaging target cells with relatively mediumor low levels of target antigen expression. For example, FIG. 6Billustrates cytotoxic effects of XTENylated and de-XTENylatedProtease-Activated T-cell engagers (PATs) on a cancer cell line with lowlevel of HER2 expression, MCF-7, where masking with XTEN polypeptidesreduced T cell-mediated cytotoxicity of the tested HER2-XPAT byapproximately 10⁴-fold. As another example, cytotoxicity of theXTENylated (masked) HER2-XPAT and the corresponding de-XTENylated(unmasked, activated) HER2-PAT against MDA-MB-453, another cancer cellline with a medium level of HER2 expression, was determined (FIG. 6C).

FIGS. 7A-7D illustrate proteolytic activation of XTENylated bispecificsin subjects and robust tumor regressions induced thereby. FIG. 7Aillustrates comparable efficacy induced with equimolar dosing of acleavable XTENylated HER2 T-cell engager (“HER2-XPAT,” indicated ashexagons) and a corresponding unmasked HER2 T-cell engager (“HER2-PAT,”indicated as triangles) in (e.g., BT-474) tumor-bearing mice. (**indicates p<0.01.) Notably, between the two tested XTENylatedbispecifics, tumor regression was observed on the cleavable XTENylatedconstruct (indicated as hexagons) but not on the non-cleavableXTENylated counterpart (indicated as diamonds), indicating thatproteolytic unmasking (de-XTENylation) is a prerequisite for efficacy.FIG. 7B illustrates efficacy of cleavable XTENylated bispecific (e.g.,HER2-XPAT) against large tumors with a single dose (e.g., 2.1 milligramsper kilograms (mpk)). The non-cleavable constructs used in theexperiments are identical to the corresponding cleavable constructs, butthe release site has been replaced with a non-cleavable sequence ofsimilar length made from GASTEP amino acids (glycine, alanine, serine,threonine, glutamate, and/or proline). FIG. 7C shows efficacy of themasked HER2-XPAT (solid triangles) at two different concentrations (15nmol/Kg and 36 nmol/Kg), compared to unmasked HER2-PAT (solid squares)and non-cleavable XPAT (solid diamonds). The data for the tumors treatedwith vehicle and vehicle+PBMCs is also shown. FIG. 7D shows thepercentage cleavage of HER2-PAT in vivo in BT-474 tumor bearing mice.

FIG. 8 illustrates lymphocyte margination induced in subjects byadministration of XTENylated HER2-XPATs. For example, upon a single-doseintravenous infusion (e.g., 25 mg/kg at a dose volume of 10 ml/kg) of anXTENylated, HER2-XPAT (e.g., set forth in Table D), a decrease inlymphocyte count (hematology) was observed, in both female and malemonkey subjects (as indicated by triangles and squares, respectively),starting at 6 hours post-dose and lasting till at least 24-72 hourspost-dose.

FIGS. 9A-9B. FIG. 9A illustrates stability of XTENylatedProtease-Activated T-cell engagers (PATs) in plasma circulation insubjects (e.g., cynomolgus monkey) (e.g., at 25 mg/kg dose). Lack ofsignificant increase in XTENylated PAT clearance relative to itsnon-cleavable form indicates minimal cleavage in periphery. Comparablepharmacokinetics were observed between the tested cleavable HER2-XPAT(solid triangle and solid square) and the non-cleavable counterpart(unfilled triangle and unfilled square). The non-cleavable constructsused in the experiments are identical to the corresponding cleavableconstructs, but the release site has been replaced with a non-cleavablesequence of similar length made from GASTEP amino acids (glycine,alanine, serine, threonine, glutamate, and/or proline). FIG. 9B showsthat there is a low frequency of HER2-XPAT proteolytic metabolites incirculation even 96 hours post administration.

FIGS. 10A-10C. FIG. 10A shows a single dose, single subject HER2-XPATdose escalation and demonstrates that all doses up to 42 mg/kg weretolerated. FIG. 10B shows a single subject HER2-PAT de-escalation schemein which the maximum tolerated dose of the unmasked HER2-PAT is 0.2mg/kg. FIG. 10C shows the plasma concentrations of masked HER2-PAT (at a450-fold higher tolerated Cmax) as compared to unmasked HER2-PAT.

FIGS. 11A-11E. FIGS. 11A and 11B show peripheral T cell Activation datathat show there is no peripheral T cell activation in response toHER2-XPAT as compared to HER2-PAT. FIGS. 11C-11E show the levels ofcytokines IL-6 (FIG. 11C), TNF-α (FIG. 11D) and IFN-γ (FIG. 11E) insubjects treated with HER2-XPAT and HER2-PAT at varying concentrations,demonstrating that HER2-XPAT does not induce cytokine release even at 50mg/kg. Note, that the normal range for cytokine levels: IL-6≤6 pg/ml,TNFα 1-10 pg/ml, IFNγ≤10 pg/ml.

FIG. 12 . AMX-818 Incubated ex vivo in plasma samples from NHPs andhumans showed minimal cleavage to unmasked TCE, even under inflammatoryconditions. AMX-818 with a fluorescent label (DyL650) attached to thetandem scFv was incubated in the indicated plasma samples for seven daysat 37° C. Samples were then run on a gel and metabolites similar in sizeto the unmasked, active form of AMX-818 were quantified using a LI-CORdetector. Inflammatory disease human samples were derived from patientswith rheumatoid arthritis, lupus, inflammatory bowel disease, andmultiple sclerosis. Cancer human samples were derived from patients withlung, breast, and colon tumors. Sample sizes: Healthy NHP N=4, HealthyHuman N=4, “Inflamed” NHP N=6, “Inflamed” Human N=27, Cancer Human N=11.

FIG. 13A-FIG. 13B. Provides additional data in support of the safetyprofile of a preferred HER2-XPAT of the present invention. The data inFIG. 13A shows a comparable PK between HER2-XPAT and the non-cleavableHER2-PAT formats, demonstrating that the protease release site remainslargely stable in circulation of cynomolgus monkeys even at high doses.FIG. 13B shows that even at high dose of HER2 XPAT there is very limitedsystemic accumulation of metabolites lacking one or both XTEN masks.

FIGS. 14A-14D. AMX818 and 818-PAT Induce Surface Expression of PD-1 on Tcells in Response to SKOV3 Tumor Cells. Surface PD-1 expression wasevaluated on CD4+ and CD8+ T cells by flow cytometry following a 48-hourco-incubation of PBMCs and SKOV3 cells at a 5:1 Effector:Target ratiowith test articles at the indicated concentrations. FIG. 14A and FIG.14C shows surface PD1 expression on CD4+ T cells in the presence ofAMX818 and 818-PAT. FIG. 14B and FIG. 14D shows surface PD1 expressionon CD8+ T cells in the presence of AMX818 and 818-PAT.

FIG. 13A-13C. AMX818 and 818-PAT Induce Surface Expression of PD-L1 on Tcells in Response to SKOV3 Tumor Cells. FIG. 15A and FIG. 15B show PD-L1expression on SKOV3 cells at a 5:1 Effector:Target ratio with testarticles at the indicated concentrations. FIG. 15C shows surface HER2expression on SKOV3 tumor cells.

FIG. 16A-16C. XPATs Are Preferentially Cleaved to Unmasked TCEs in HumanTumors Implanted in Living Mice, with Minimal Cleavage Observed inHealthy Tissues. A single dose of 1.8 mg/kg (13 nM) redfluorophore-labeled (Alexa) XPAT was injected into mice implanted withhuman tumors (FIG. 16A). Two days later, tumors and healthy organs wereharvested to measure protease cleavage in vivo (results shown in FIG.16B). The green-fluorophore-labeled (Dyl800) XPAT was added post-tissueharvesting in the presence of protease inhibitors to account forartifactual cleavage that could result from release of non-relevantintracellular proteases during the processing. By comparing thegenerated cleavage products of both red and green fluorophore-labeledXPATs, we can determine the cleavage that occurred in the varioustissues in vivo vs artifactually during tissue processing. On average20% of XPAT in the tumor is activated by Day 2 after injection into miceimplanted with tumors (n=31 across 9 tumor types; FIG. 16C).

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

There is a significant unmet need in cancer therapeutics. While TCEshave been shown to be effective in inducing remission in certaincancers, they have not produced widespread therapeutics due to theirextreme potency and on target, off tumor toxicities in healthy tissues.By way of explanation, the TCEs form a bridge between T cells and tumorcells and activate T cell-mediated of the tumor cell and furtherinitiating a cytokine amplification cascades that promotes furtherkilling and potentially provides long term immunity. T cells activatedby TCEs to release cytolytic perforin/granzymes in a manner that isindependent of antigen-MHC recognition. This creates a two-foldresponse: direct tumor cell death and amplification of tumor killingthrough initiation of a powerful cytokine response from the tumor cells.The direct tumor cell death results in release of tumor antigens. Thecytokine response includes, among others, increased interferon-γ whichstimulates CD8 T cell activity and stimulates antigen presentation byAPCs; increased IL2 which causes increased proliferation of activatedT-cells, and increased CXCL9 and 10 response which increases T cellrecruitment. Together the release of tumor antigens and the initiationof the cytokine response results in activation of the endogenous T-cellresponse which potentially cases epitope spreading to induce long termimmunity.

The toxicity challenge with TCEs arises out the fact that most tumortargets are, to some extent, also expressed in healthy tissue, andnormal cells also can produce the cytokines response resulting incytokine release syndrome (CRS). These two powerful responses of healthtissue to T cell activation by TCEs results in an overall lacktherapeutic index for these agents.

The present invention overcomes the drawbacks in the existing TCEs byproviding a conditionally-activated TCE, XPAT or XTENylatedProtease-Activated bispecific T Cell Engager targeting HER2 (referred toherein as HER2-XPAT, and exemplified as AMX818). More particularly, theXPAT of the present invention exploits the dysregulated proteaseactivity present in tumors vs. healthy tissues, enabling expansion ofthe therapeutic index. The XPAT core consists of 2 single chain antibodyfragments (scFvs) targeting CD3 and the tumor target (in exemplaryembodiments the tumor target is HER2). Two unstructured polypeptidemasks (XTENs) are attached to the core that sterically reduce targetengagement of either the tumor target and/or CD3 and extend proteinhalf-life. The properties of the XTEN polymer also minimize thepotential for immunogenicity, as its lack of stable tertiary structuredisfavors antibody binding and the absence of hydrophobic, aromatic andpositively charged residues that serve as anchor residues for peptideMHC II binding reduces the potential for T cell epitopes. In humans,minimal immunogenicity of XTEN polymers was observed in >200 patientstreated with drugs containing XTEN in the context of half-life extendedforms of human growth hormone and Factor VIII. Protease cleavage sitesat the base of the XTEN masks enable proteolytic activation of XPAT inthe tumor microenvironment, unleashing a small, highly potent TCE thatis capable redirecting cytotoxic T cells to kill target-expressing tumorcells. In healthy tissues, where protease activity is tightly regulated,XPATs should remain predominantly inactive as intact prodrugs, thusexpanding the therapeutic index compared to unmasked TCEs.

In addition to localized activation, the short half-life of the unmaskedPAT form should further widen the therapeutic index while providing thepotency of T-cell immunity to improve the eradication of solid tumors.The release sites used in the XPATs can be cleaved across a broad arrayof tumors by proteases that are collectively involved in every cancerhallmark (growth; survival and death; angiogenesis; invasion andmetastasis; inflammation; and immune evasion). Thus, TCE activity of theXPATs is localized to tumors by exploiting the enhanced proteaseactivity that is upregulated in all stages of cancer and tumordevelopment but is tightly regulated in healthy tissues.

The components of the exemplary HER2-XPAT, AMX-818, have been optimizedto achieve the desired balance between providing sufficient protectionin healthy tissue while retaining the necessary potency in tumors acrossa broad range of cancers. To reduce the potential for T cell activationby the prodrug, a lower binding affinity was selected for the a-CD3domain in addition to a longer XTEN polymer mask (576 amino acid mask vs256 amino acids on the HER2 side). To ensure sufficient activation ofAMX-818 in the tumor, the protease release site at the base of the XTENmasks was engineered to be cleaved by at least 8 different proteasesamong 3 different classes that have been reported to be over-expressedor dysregulated in cancer; these include several matrixmetalloproteinases (MMPs), Matriptase, uPA, and the cysteine protease,legumain. As a safety checkpoint, co-engagement of both CD3 and HER2 byAMX-818 is required for T cell activation. Activation of T cells shouldnot occur if AMX-818 is unmasked in inflamed tissues where HER2expression is absent or if it encounters HER2 expressed in healthytissue where proteases are tightly controlled. This AND-gate feature isexpected to provide preferential activation in the tumor where bothelevated protease activity and high HER2 expression are present.

Thus presence of the XTEN on the XPAT produces an agent that a longhalf-life, weak target engagement and negligible T-cell activation. Oncethe XTEN is removed by the action of the proteases in the tumormicroenvironment, this preferential activation of the XPATs produces anactivated drug (PAT, without the XTEN) that has a short half-life,optimal target engagement, and highly efficient T-cell activation,thereby producing a powerful activated drug with an enhanced therapeuticindex. The HER2-XPATs of the present invention a capable of improvingthe toxicity profile of T cell engagers while maintaining their potencyagainst solid tumors, thus enabling a significant increase in thetherapeutic index and expansion of target landscape for this potentmodality.

Summary of Data Generated from AMX-818, an Exemplary HER2-XPAT

Target binding and in vitro biological activities of AMX-818 have beencharacterized in multiple studies. Equilibrium binding analysisutilizing surface plasmon resonance demonstrated highly comparableaffinities for AMX-818 and its metabolites between human and cynomolgusmonkey HER2 and CD3, supporting the use of cynomolgus monkey as aspecies for toxicity and PK studies. Proteolytically-activatedAMX-818(PAT) bound to human and cynomolgus monkey HER2 with 2.4 nM and2.0 nM affinities, respectively, and to human and cynomolgus monkey CD3with 26.3 nM and 21.5 nM affinities, respectively. Masking of AMX-818reduced its affinities to HER2 by 10-fold and to CD3 by approximately6-fold for both species. AMX-818 bound to human and cynomolgus monkeyHER2 with 24.9 nM and 20.1 nM affinities, while CD3 affinities for humanand cynomolgus monkey were 160 nM and 140.3 nM, respectively.

The activity of a TCE depends on its ability to activate T cells througheffective stimulation of the T cell receptor (TCR). The extreme potencyof TCEs derives from the minimal requirement for as few as 3 TCRs tobecome stimulated and coalesce to form an immune synapse between the Tcell and target cell to initiate cytotoxicity. While T-cell engagers areprimarily known for inducing cytotoxicity, their potency also involvescytokine-driven actions downstream of T-cell activation that enhance andamplify the anti-tumor immune response. T-cell activation by AMX-818,its prototype AMX-818-P1, and its proteolytic metabolites wascharacterized in vitro utilizing a Jurkat NFAT-reporter cell and primaryhuman PBMCs in the presence of HER2-high-expressing tumor cells, BT-474(breast) and SKOV-3 (ovarian). Human T cells were also assessed forupregulation of the surface activation marker CD69 and the inhibitoryreceptor PD-1 by flow cytometry. As an indirect measure of T-cellactivation, upregulation of the PD-1 ligand PD-L1 was assessed on thesurface of SKOV3 tumor targets, as it is induced in response to IFN-γsecreted by activated T cells.

AMX-818(PAT) activated the Jurkat NFAT-Luciferase reporter T cells inthe presence of BT-474 cells with EC₅₀ values in the 70 pM range whileresponse to the masked AMX-818 and AMX-818-P1 was significantlyattenuated by 4 orders of magnitude, with maximal responses reduced by80-90% compared to that of the activated AMX-818(PAT). T-cell activationby AMX-818(PAT) was not observed in the absence of HER2+BT-474 tumorcells, demonstrating that monovalent engagement of CD3 was notsufficient for activation and that co-engagement of both CD3 and tumortarget were required for effective T cell receptor (TCR) stimulation.The singly masked AMX-818 metabolites, AMX-818(1x-C), and AMX-818(1x-N)demonstrated intermediate activity. AMX-818-NoClvSite induced nodetectable T-cell activation, suggesting that the minimal responseobserved by the AMX-818 was likely driven by proteolytic cleavage.

AMX-818(PAT) and prodrug AMX-818 induced CD69 and PD-1 expression on thesurface of both CD4+ and CD8+ T-cell subsets and PD-L1 on SKOV3 tumorcells to comparable degrees. However, the dose-response curve forAMX-818 was shifted on average 400 to 650-fold higher relative to thatof AMX-818(PAT), further demonstrating effective functional masking ofthe XTEN masks on AMX-818.

AMX-818 and its metabolites were characterized for their cytotoxicactivity and induction of inflammatory cytokines. Peripheral bloodmononuclear cells (PBMCs) were used as effector cells and the HER2-highBT-747 breast tumor line selected as the tumor target cell. Becausecardiac tissues are known to express HER2 and, while rare, cardiactoxicities have been observed in patients treated with someHER2-targeted therapies, primary cardiomyocytes (HER2 low to medium)were selected to represent a more physiologic cytolytic target forAMX-818 and its proteolytic metabolites. A luminescence-basedcytotoxicity assay was conducted at a 1:1 effector:target cell ratio andcell free supernatants were collected for measurement of TCE-inducedcytokines.

AMX-818(PAT) demonstrated highly potent cytotoxicity against BT-474tumor cells, showing nearly complete target cell killing withhalf-maximal inhibitory concentration (IC₅₀) values in the 5-11 pMrange. With lower HER2-expressing human cardiomyocytes as targets—cellsnot expected to exhibit dysregulated protease activity—cytotoxicityresponses were reduced by approximately 13-fold and maximal killing wasincomplete, approaching only 50-60%. The cytotoxic response by AMX-818against both BT-474 and cardiomyocyte cells was strongly attenuated,with average IC₅₀ values shifted by 2500- to 3000-fold, demonstratingeffective functional masking of the prodrug by its XTEN polymer masks.The masks provide synergistic protection in cytotoxicity well abovetheir combined impact on reducing target binding by impeding formationof the functional immune synapse that is required to initiate targetcell killing. AMX-818 and its prototype AMX-818-P1 showed comparablecytotoxicity consistent with their nearly identical composition.Cytotoxicity of the singly masked proteolytic metabolites, AMX-818(1x-N)and AMX-818(1x-C) was intermediate between AMX-818(PAT) and AMX-818,demonstrating partial protection by a single mask. The cytotoxicityobserved by AMX-818 was likely due to proteolytic cleavage based on thefurther reduction in activity provided by AMX-818-NoClvSite, the formatlacking both protease cleavage sites.

In general, the relative potencies of AMX-818 and its metabolites ininduction of cytokine secretion in supernatants from cytotoxicity assaysmirrored those observed in cytotoxicity to both BT-474 and cardiomyocytetarget cells (where AMX-818(PAT) was most potent>singly masked forms>andAMX-818 least potent). The cytokine IC₅₀ values were on average higherthan those for the cytotoxic response demonstrating that cytotoxicity isthe more sensitive assay among the two. Responses to AMX-818 in thepresence of both BT-474 and cardiomyocytes were reduced by severalorders of magnitude relative to those of AMX-818(PAT), while the singlymasked metabolites AMX-818(1x-N) and AMX-818(1x-C) showed intermediateresponse.

In the presence of cardiomyocytes, the maximal levels of cytokinesinduced by AMX-818 at its highest concentration tested (300 nM) weremarkedly reduced relative to those induced by AMX-818(PAT), with theexception of IL-6. In the presence of both BT-474 and cardiomyocytes,elevated IL-6 levels were detected at 300 nM concentrations of bothAMX-818 and AMX-818-NoClvSite, that in most cases exceeded the maximallevels produced by AMX-818(PAT) by 2- to 6-fold. Of note, this is incontrast to what is observed in vivo in cynomolgus monkeys, where peaksystemic levels of IL-6 are >9-fold higher at the 0.2 mg/kg MTD ofAMX-818(PAT) than at the 42 mg/kg MTD of AMX-818.

In assessing the induction of cytokines in the absence ofHER2-expressing target cells, cytokines IL-2, IL-4, TNF-α, and IFN-γwere not induced in human PBMC cultures treated with suspension andplate-coated AMX-818 or AMX-818(PAT). At its highest concentrationtested (500 nM), soluble AMX-818 induced low levels of IL-10 from allPBMC donors. IL-6 was a notable exception, where in the soluble formatat 500 nM, AMX-818 induced levels of IL-6 from all donors that exceededthose induced by the anti-CD3 antibody positive control by an average of4.6-fold increase in the means. High levels of IL-6 were also induced by2 of 5 donors in the wet plate-coated format, and lower levels of IL-6were also seen at the highest concentration of AMX-818(PAT). However,importantly, such elevated IL-6 levels were not accompanied by increasesin TNF-α, IFN-γ, and IL-2, which are cytokines commonly co-associatedwith IL-6 secretion under conditions of CRS, nor were they observed incynomolgus monkeys dosed with AMX-818 at up to 50 mg/kg. In contrast,the activated AMX-818(PAT) induced high levels of IL-6 in cynos at doses≤0.3 mg/kg that were accompanied by increases in the additionalinflammatory cytokines.

In vivo pharmacology, PK, and toxicology studies were conducted tocharacterize the efficacy and safety of AMX-818 and its metabolites. Inaddition to standard toxicology endpoints, the proteolytic stability ofAMX-818 in circulation was evaluated in cynomolgus monkeys administeredhigh doses of AMX-818 or under conditions of induced inflammation and invitro following extended incubation in plasma from patients with canceror systemic autoimmune disease. Preferential cleavage of AMX-818 intumors was evident resulting in protease-dependent efficacy, while itsperipheral stability in NHP provided a large safety margin relative toAMX-818(PAT) that predicts an increased therapeutic index. Together,these data support strong conditional masking of AMX818 that enableslocalized tumor activity with concomitant stability and effectivemasking in circulation and peripheral tissues.

Several in vivo efficacy studies were conducted to evaluate the impactof AMX-818 in redirecting T cells to kill HER2-expressing tumors. SinceAMX-818 is not cross-reactive to mouse HER2 or CD3, immunodeficient micewere inoculated with human HER2-expressing xenograft tumors andengrafted with human PBMC (hPBMC) as a source of effector T cells.Anti-tumor activity of AMX-818 was assessed in the HER2-high BT-474breast model (˜975,000 HER2 receptors) and HER2-low HT-55 colorectalmodel (25,000 receptors).

In the BT474 model, administration of equimolar doses of AMX-818 and itsprototype AMX-818-P1 at 2.1 mg/kg or the unmasked AMX-818(PAT) at 0.9mg/kg induced robust and complete tumor regressions. The anti-tumorefficacy of AMX-818 was dependent on protease cleavage of its masks, asdemonstrated by the lack of significant tumor growth inhibition in micetreated with a form of AMX-818 lacking its protease release sites(AMX-818-NoClvSite). AMX-818-P1 and AMX-818(PAT) induced comparableactivation of T cells in the tumor microenvironment as assessed byupregulation of activation markers CD25 and CD69 on CD4 and CD8+ T cellsby flow cytometry. Importantly, T cells were not activated in theperiphery even by the unmasked AMX-818(PAT) where human HER2 is notexpressed, consistent with a requirement for dual engagement of bothHER2 and CD3 to activate T cells and redirect their killing.

A single 2.1 mg/kg dose of AMX-818 was sufficient to induce tumorregressions in mice bearing large established BT-474 tumors (478 mm³mean tumor volume) within 4 days of dosing. The efficacy was dependenton both HER2 expression and T cells, as demonstrated by the lack ofactivity of a non-tumor binding variant NB-XPAT (a version of AMX-818,in which its HER2 binding domain was replaced with a non-HER2 bindingscFv) or of AMX-818 when dosed in tumor-bearing mice lacking hPBMCs.These findings further support the requirement for dual engagement ofAMX-818 for its activity and provide a safety measure should AMX-818become cleaved in normal tissue where HER2 is absent.

In mice bearing HER2-low HT-55 tumors, AMX-818 also induced dose- andprotease-dependent efficacy, with 5.1 mg/kg inducing complete tumorregression in all mice (103% TGI, p<0.01), and 2.1 mg/kg inducing 70%tumor growth inhibition. Efficacy by AMX-818 in a model expressing25,000 HER2 receptors provides potential for treating patients withmultiple cancer types that include tumors with low levels of HER2expression. Finally, preferential unmasking of a fluorophore-labeledAMX-818 to an average of 25.2% AMX-818(PAT) was evident in tumorscompared to <2% in heart, brain and liver tissue combined following a2-day incubation in BT-474 tumor-bearing mice, supporting the keytenants of localized dysregulation of proteases in tumors and thedominance of protease inhibition in normal tissues.

Terminology

As used herein, the following terms have the meanings ascribed to themunless specified otherwise.

As used in the specification and claims, the singular forms “a,” “an,”and “the” include plural references unless the context clearly dictatesotherwise. For example, the term “a cell” includes a plurality of cells,including mixtures thereof.

The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably herein to refer to polymers of amino acids of anylength. The polymer may be linear or branched, it may comprise modifiedamino acids, and it may be interrupted by non-amino acids. The termsalso encompass an amino acid polymer that has been modified, forexample, by disulfide bond formation, glycosylation, lipidation,acetylation, phosphorylation, or any other manipulation, such asconjugation with a labeling component.

As used herein the term “amino acid” refers to either natural and/orunnatural or synthetic amino acids, including but not limited to glycineand both the D or L optical isomers, and amino acid analogs andpeptidomimetics. Standard single or three letter codes are used todesignate amino acids.

A “host cell” includes an individual cell or cell culture which can beor has been a recipient for the subject vectors. Host cells includeprogeny of a single host cell. The progeny may not necessarily becompletely identical (in morphology or in genomic of total DNAcomplement) to the original parent cell due to naturally occurring orgenetically engineered variation

A “chimeric” protein contains at least one fusion polypeptide comprisingregions in a different position in the sequence than that which occursin nature. The regions may normally exist in separate proteins and arebrought together in the fusion polypeptide; or they may normally existin the same protein but are placed in a new arrangement in the fusionpolypeptide. A chimeric protein may be created, for example, by chemicalsynthesis, or by creating and translating a polynucleotide in which thepeptide regions are encoded in the desired relationship.

“Conjugated”, “linked,” “fused,” and “fusion” are used interchangeablyherein. These terms refer to the joining together of two more chemicalelements or components, by whatever means including chemical conjugationor recombinant means.

The terms “polynucleotides”, “nucleic acids”, “nucleotides” and“oligonucleotides” are used interchangeably. They refer to a polymericform of nucleotides of any length, either deoxyribonucleotides orribonucleotides, or analogs thereof. Polynucleotides may have anythree-dimensional structure, and may perform any function, known orunknown. A polynucleotide may comprise modified nucleotides, such asmethylated nucleotides and nucleotide analogs. If present, modificationsto the nucleotide structure may be imparted before or after assembly ofthe polymer. The sequence of nucleotides may be interrupted bynon-nucleotide components. A polynucleotide may be further modifiedafter polymerization, such as by conjugation with a labeling component.

As used herein, polynucleotides having “homology” or that are“homologous” are those which hybridize under stringent conditions asdefined herein and have at least 70%, preferably at least 80%, morepreferably at least 90%, more preferably 95%, more preferably 97%, morepreferably 98%, and even more preferably 99% sequence identity to thosesequences.

The terms “percent identity” and “% identity,” as applied topolynucleotide sequences, refer to the percentage of residue matchesbetween at least two polynucleotide sequences aligned using astandardized algorithm. Such an algorithm may insert, in a standardizedand reproducible way, gaps in the sequences being compared in order tooptimize alignment between two sequences, and therefore achieve a moremeaningful comparison of the two sequences. Percent identity may bemeasured over the length of an entire defined polynucleotide sequence,for example, as defined by a particular SEQ ID number, or may bemeasured over a shorter length, for example, over the length of afragment taken from a larger, defined polynucleotide sequence, forinstance, a fragment of at least 45, at least 60, at least 90, at least120, at least 150, at least 210 or at least 450 contiguous residues.Such lengths are exemplary only, and it is understood that any fragmentlength supported by the sequences shown herein, in the tables, figuresor Sequence Listing, may be used to describe a length over whichpercentage identity may be measured.

“Percent (%) amino acid sequence identity,” with respect to thepolypeptide sequences identified herein, is defined as the percentage ofamino acid residues in a query sequence that are identical with theamino acid residues of a second, reference polypeptide sequence or aportion thereof, after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent sequence identity, and notconsidering any conservative substitutions as part of the sequenceidentity. Alignment for purposes of determining percent amino acidsequence identity can be achieved in various ways that are within theskill in the art, for instance, using publicly available computersoftware such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software.Those skilled in the art can determine appropriate parameters formeasuring alignment, including any algorithms needed to achieve maximalalignment over the full length of the sequences being compared. Percentidentity may be measured over the length of an entire definedpolypeptide sequence, for example, as defined by a particular SEQ IDnumber, or may be measured over a shorter length, for example, over thelength of a fragment taken from a larger, defined polypeptide sequence,for instance, a fragment of at least 15, at least 20, at least 30, atleast 40, at least 50, at least 70 or at least 150 contiguous residues.Such lengths are exemplary only, and it is understood that any fragmentlength supported by the sequences shown herein, in the tables, figuresor Sequence Listing, may be used to describe a length over whichpercentage identity may be measured.

As used herein, “repetitiveness” of an XTEN sequence refers to the 3-merrepetitiveness and can be measured by computer programs or algorithms orby other means known in the art. The 3-mer repetitiveness of an XTEN isassessed by determining the number of occurrences of the overlapping3-mer sequences within the polypeptide. For example, a polypeptide of200 amino acid residues has 198 overlapping 3-amino acid sequences(3-mers), but the number of unique 3-mer sequences will depend on theamount of repetitiveness within the sequence. A score can be generated(hereinafter “subsequence score”) that is reflective of the degree ofrepetitiveness of the 3-mers in the overall polypeptide sequence. In thecontext of the present invention, “subsequence score” means the sum ofoccurrences of each unique 3-mer frame across a 200 consecutive aminoacid sequence of the polypeptide divided by the absolute number ofunique 3-mer subsequences within the 200 amino acid sequence. Examplesof such subsequence scores derived from the first 200 amino acids ofrepetitive and non-repetitive polypeptides are presented in Example 73of International Patent Application Publication No. WO 2010/091122 A1,which is incorporated by reference in its entirety. In some embodiments,the present invention provides BPXTEN each comprising XTEN in which theXTEN can have a subsequence score less than 16, or less than 14, or lessthan 12, or more preferably less than 10.

The term “substantially non-repetitive XTEN,” as used herein, refers toan XTEN, wherein (1) there are few or no instances of four contiguousamino acids in the XTEN sequence that are identical amino acid types andwherein (2) the XTEN has a subsequence score (defined in the precedingparagraph herein) of 12, or 10 or less or that there isn't a pattern inthe order, from N- to C-terminus, of the sequence motifs that constitutethe polypeptide sequence.

A “vector” is a nucleic acid molecule, preferably self-replicating in anappropriate host, which transfers an inserted nucleic acid molecule intoand/or between host cells. The term includes vectors that functionprimarily for insertion of DNA or RNA into a cell, replication ofvectors that function primarily for the replication of DNA or RNA, andexpression vectors that function for transcription and/or translation ofthe DNA or RNA. Also included are vectors that provide more than one ofthe above functions. An “expression vector” is a polynucleotide which,when introduced into an appropriate host cell, can be transcribed andtranslated into a polypeptide(s). An “expression system” usuallyconnotes a suitable host cell comprised of an expression vector that canfunction to yield a desired expression product.

The term “t_(1/2)” as used herein means the terminal half-lifecalculated as ln(2)/K_(e1). K_(e1) is the terminal elimination rateconstant calculated by linear regression of the terminal linear portionof the log concentration vs. time curve. Half-life typically refers tothe time required for half the quantity of an administered substancedeposited in a living organism to be metabolized or eliminated by normalbiological processes. The terms “t_(1/2)”, “terminal half-life”,“elimination half-life” and “circulating half-life” are usedinterchangeably herein.

The terms “antigen,” “target antigen,” or “immunogen” are usedinterchangeably herein to refer to the structure or binding determinantthat an antibody fragment or an antibody fragment-based therapeuticbinds to or has specificity against.

The term “payload” as used herein refers to a protein or peptidesequence that has biological or therapeutic activity; the counterpart tothe pharmacophore of small molecules. Examples of payloads include, butare not limited to, cytokines, enzymes, hormones and blood and growthfactors. Payloads can further comprise genetically fused or chemicallyconjugated moieties such as chemotherapeutic agents, antiviralcompounds, toxins, or contrast agents. These conjugated moieties can bejoined to the rest of the polypeptide via a linker which may becleavable or non-cleavable.

As used herein, “treatment” or “treating,” “palliating,” and“ameliorating” are used interchangeably herein. These terms refer to anapproach for obtaining beneficial or desired results including but notlimited to a therapeutic benefit and/or a prophylactic benefit. By“therapeutic benefit” is meant eradication or amelioration of theunderlying disorder being treated. Also, a therapeutic benefit isachieved with the eradication or amelioration of one or more of thephysiological symptoms associated with the underlying disease conditionsuch that an improvement is observed in the subject, notwithstandingthat the subject may still be afflicted with the underlying disorder.For prophylactic benefit, the compositions may be administered to asubject at risk of developing a particular disease condition, or to asubject reporting one or more of the physiological symptoms of adisease, even though a diagnosis of this disease may not have been made.

A “therapeutic effect,” as used herein, refers to a physiologic effect,including but not limited to the cure, mitigation, amelioration, orprevention of disease condition in humans or other animals, or tootherwise enhance physical or mental wellbeing of humans or animals,caused by a fusion polypeptide of the invention other than the abilityto induce the production of an antibody against an antigenic epitopepossessed by the biologically active protein. Determination of atherapeutically effective amount is well within the capability of thoseskilled in the art, especially in light of the detailed disclosureprovided herein.

The terms “therapeutically effective amount” and “therapeuticallyeffective dose,” as used herein, refers to an amount of a biologicallyactive protein, either alone or as a part of a fusion proteincomposition, that is capable of having any detectable, beneficial effecton any symptom, aspect, measured parameter or characteristics of adisease state or condition when administered in one or repeated doses toa subject. Such effect need not be absolute to be beneficial. Thedisease condition can refer to a disorder or a disease.

The term “therapeutically effective dose regimen,” as used herein,refers to a schedule for consecutively administered doses of abiologically active protein, either alone or as a part of a fusionprotein composition, wherein the doses are given in therapeuticallyeffective amounts to result in sustained beneficial effect on anysymptom, aspect, measured parameter or characteristics of a diseasestate or condition.

Fusion Polypeptide

Disclosed herein includes a polypeptide (or fusion polypeptide)comprising one or more extended recombinant polypeptides (XTEN or XTENs)(as described more fully hereinbelow), a bispecific antibody construct(BsAb) linked to the XTEN(s), and one or more release segments (RS); therelease segment is positioned between the XTEN and the bispecificantibody construct (BsAb); and the polypeptide (or fusion polypeptide)has an N-terminal amino acid and a C-terminal amino acid.

In some embodiments, the polypeptide comprises a first XTEN (such asthose described below in the “EXTENDED RECOMBINANT POLYPEPTIDE (XTEN)”section or described anywhere else herein). In some embodiments, thepolypeptide further comprises a second XTEN (such as those describedbelow in the “EXTENDED RECOMBINANT POLYPEPTIDE (XTEN)” section ordescribed anywhere else herein). In some embodiments, the polypeptidecomprises an XTEN at or near its N-terminus (an “N-terminal XTEN”). Insome embodiments, the polypeptide comprises an XTEN at or near itsC-terminus (a “C-terminal XTEN”). In some embodiments, the polypeptidecomprises both an N-terminal XTEN and a C-terminal XTEN. In someembodiments, the first XTEN is an N-terminal XTEN and the second XTEN isa C-terminal XTEN. In some embodiments, the first XTEN is a C-terminalXTEN and the second XTEN is an N-terminal XTEN.

As the bispecific antibody (BsAb), a biologically active polypeptide(“BP”), is linked to the one or more XTENs within the polypeptide, thepolypeptide may be referred to as an XTEN-containing fusion polypeptide:“BPXTEN.”

The XTEN can comprise one or more barcode fragments (as described morefully below) releasable (configured to be released) from the XTEN upondigestion of the fusion polypeptide (or BPXTEN) by a protease. In someembodiments, each barcode fragment differs in sequence and molecularweight from all other peptide fragments (including all other barcodefragments if present) that are releasable from the polypeptide uponcomplete digestion of the polypeptide by the protease.

The (fusion) polypeptide can comprise one or more reference fragments(as described more fully below) releasable (configured to be released)from the polypeptide, for example, upon the protease digestion whichreleases the barcode fragment(s) from the polypeptide. In someembodiments, each reference fragment can be a single reference fragmentthat differs in sequence and molecular weight from all other peptidefragments that are releasable from the polypeptide upon digestion of thepolypeptide by the protease.

In some embodiments, the polypeptide has an N-terminal amino acid and aC-terminal amino acid. The polypeptide can comprise (a) an extendedrecombinant polypeptide (XTEN) comprising a barcode fragment (BAR)releasable from the polypeptide upon digestion by a protease; (b) abispecific antibody construct (BsAb), comprising a first antigen bindingfragment (AF1) that specifically binds to cluster of differentiation 3 Tcell receptor (CD3), which AF1 comprises light chaincomplementarity-determining regions 1 (CDR-L1), 2 (CDR-L2), and 3(CDR-L3) and heavy chain complementarity-determining regions 1 (CDR-H1),2 (CDR-H2), and 3 (CDR-H3), wherein the CDR-H3 comprises an amino acidsequence of SEQ ID NO:10; and a second antigen binding fragment (AF2)that specifically binds to human epidermal growth factor receptor 2(HER2); and (c) a release segment (RS) positioned between the XTEN andthe bispecific antibody construct, wherein the XTEN is characterized inthat: (i) it comprises at least 100, or at least 150 amino acids; (ii)at least 90% of its amino acid residues are identified herein by glycine(G), alanine (A), serine (S), threonine (T), glutamate (E) or proline(P); (iii) it comprises at least 4 different types of amino acidsidentified herein by G, A, S, T, E, or P; and (iv) the XTEN is formedfrom a plurality of non-overlapping sequence motifs that are each from 9to 14 amino acids in length, wherein the plurality of non-overlappingsequence motifs comprise: (1) a set of non-overlapping sequence motifs,wherein each non-overlapping sequence motif of the set ofnon-overlapping sequence motifs is repeated at least two times in theXTEN; and (2) a non-overlapping sequence motif that occurs only oncewithin the XTEN; wherein the barcode fragment (BAR) includes at leastpart of the non-overlapping sequence motif that occurs only once withinthe XTEN; wherein the barcode fragment (BAR) differs in sequence andmolecular weight from all other peptides fragments that are releasablefrom the polypeptide upon complete digestion of the polypeptide by theprotease; and wherein the barcode fragment (BAR) does not include theN-terminal amino acid or the C-terminal amino acid of the polypeptide.The polypeptide can be expressed as a fusion protein. The fusionprotein, in an uncleaved state, can have a structural arrangement fromN-terminus to C-terminus identified herein by AF1-AF2-RS-XTEN,AF2-AF1-RS-XTEN, XTEN-RS-AF1-AF2, or XTEN-RS-AF2-AF1.

In some embodiments of the polypeptides of this disclosure, the XTEN isa first extended recombinant polypeptide (XTEN1); the plurality ofnon-overlapping sequence motifs, from which the XTEN1 is formed, is afirst plurality of non-overlapping sequence motifs; the BAR is a firstbarcode fragment (BAR1); and the RS is a first release segment (RS1). Insome embodiments, the polypeptide can further comprise: (d) a secondextended recombinant polypeptide (XTEN2), comprising: a second barcodefragment (BAR2) releasable from the polypeptide upon digestion by theprotease; and (e) a second release segment (RS2) positioned between thesecond XTEN (XTEN2) and the bispecific antibody construct (BsAb),wherein the XTEN2 is characterized in that: (i) it comprises at least100, or at least 150 amino acids; (ii) at least 90% of its amino acidresidues are identified herein by glycine (G), alanine (A), serine (S),threonine (T), glutamate (E) or proline (P); and (iii) it comprises atleast 4 different types of amino acids identified herein by G, A, S, T,E, or P; wherein the second barcode fragment (BAR2) differs in sequenceand molecular weight from all other peptides fragments that arereleasable from the polypeptide upon complete digestion of thepolypeptide by the protease; and wherein the second barcode fragment(BAR2) does not include the N-terminal amino acid or the C-terminalamino acid of the polypeptide. The XTEN1 can be positioned N-terminal ofthe bispecific antibody construct (BsAb) and the XTEN2 can be positionedC-terminal of the bispecific antibody construct (BsAb). Alternatively,the XTEN1 can be positioned C-terminal of the bispecific antibodyconstruct (BsAb) and the XTEN2 can be positioned N-terminal of thebispecific antibody construct (BsAb). In some embodiments of thepolypeptide, where (iv) the XTEN2 can be formed from a second pluralityof non-overlapping sequence motifs that are each from 9 to 14 aminoacids in length, wherein the second plurality of non-overlappingsequence motifs comprise: (1) a second set of non-overlapping sequencemotifs, wherein each non-overlapping sequence motif of the second set ofnon-overlapping sequence motifs is repeated at least two times in thesecond XTEN; and (2) a non-overlapping sequence motif that occurs onlyonce within the second XTEN; and wherein the second barcode fragment(BAR2) includes at least part of the non-overlapping sequence motif thatoccurs only once within the second XTEN. The polypeptide can beexpressed as a fusion protein, where the fusion protein, in an uncleavedstate, can have a structural arrangement from N-terminus to C-terminusidentified herein by XTEN1-RS1-AF1-AF2-RS2-XTEN2,XTEN1-RS1-AF2-AF1-RS2-XTEN2, XTEN2-RS2-AF1-AF2-RS1-XTEN1,XTEN2-RS2-AF2-AF1-RS1-XTEN1, XTEN1-RS1-diabody-RS2-XTEN2, orXTEN2-RS2-diabody-RS1-XTEN1. The diabody can comprise a light chainvariable region (VL_(I)) of the AF1, a heavy chain variable region(VH_(I)) of the AF1, a light chain variable region (VL_(II)) of the AF2,and a heavy chain variable region (VH_(II)) of the AF2.

In some embodiments of the polypeptides of this disclosure, (a) thefirst extended recombinant polypeptide (XTEN1) can comprise an aminoacid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% sequence identity to a sequence set forth inTable 3a; (b) the bispecific antibody construct (BsAb) can comprise: (I)the first antigen binding fragment (AF1) comprising light chaincomplementarity-determining regions 1 (CDR-L1), 2 (CDR-L2), and 3(CDR-L3) and heavy chain complementarity-determining regions 1 (CDR-H1),2 (CDR-H2), and 3 (CDR-H3), wherein the CDR-H1, the CDR-H2, and theCDR-H3 comprise amino acid sequences of SEQ ID NOS: 8, 9, and 10,respectively; and (II) the second antigen binding fragment (AF2)comprising a light chain variable region (VL_(II)) identified herein bySEQ ID NOS: 778-783 and a heavy chain variable region (VH_(II))identified herein by SEQ ID NOS: 878-883; (c) the first release segment(RS1) comprises an amino acid sequence having at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to asequence identified herein by SEQ ID NOS: 7001-7626; (d) the secondextended recombinant polypeptide (XTEN2) comprises an amino acidsequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% sequence identity to a sequence set forth in Table 3a;and (e) the second release segment (RS2) comprises an amino acidsequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% sequence identity to a sequence identified herein by SEQ IDNOS: 7001-7626, where the polypeptide can have a structural arrangementfrom N-terminus to C-terminus identified herein by:XTEN1-RS1-AF2-AF1-RS2-XTEN2, XTEN1-RS1-AF1-AF2-RS2-XTEN2,XTEN2-RS2-AF2-AF1-RS1-XTEN1, or XTEN2-RS2-AF1-AF2-RS1-XTEN1.

Extended Recombinant Polypeptide (XTEN)

Chain Length and Amino Acid Composition

In some embodiments, the XTEN comprises at least 100, or at least 150amino acids. In some embodiments, the XTEN is from 100 to 3,000, or from150 to 3,000 amino acids in length. In some embodiments, the XTEN isfrom 100 to 1,000, or from 150 to 1,000 amino acids in length. In someembodiments, the XTEN is at least (about) 100, at least (about) 150, atleast (about) 200, at least (about) 250, at least (about) 300, at least(about) 350, at least (about) 400, at least (about) 450, at least(about) 500, at least (about) 550, at least (about) 600, at least(about) 650, at least (about) 700, at least (about) 750, at least(about) 800, at least (about) 850, at least (about) 900, at least(about) 950, at least (about) 1,000, at least (about) 1,100, at least(about) 1,200, at least (about) 1,300, at least (about) 1,400, at least(about) 1,500, at least (about) 1,600, at least (about) 1,700, at least(about) 1,800, at least (about) 1,900, or at least (about) 2,000 aminoacids in length. In some embodiments, the XTEN is at most (about) 100,at most (about) 150, at most (about) 200, at most (about) 250, at most(about) 300, at most (about) 350, at most (about) 400, at most (about)450, at most (about) 500, at most (about) 550, at most (about) 600, atmost (about) 650, at most (about) 700, at most (about) 750, at most(about) 800, at most (about) 850, at most (about) 900, at most (about)950, at most (about) 1,000, at most (about) 1,100, at most (about)1,200, at most (about) 1,300, at most (about) 1,400, at most (about)1,500, at most (about) 1,600, at most (about) 1,700, at most (about)1,800, at most (about) 1,900, or at most (about) 2,000 amino acids inlength. In some embodiments, the XTEN has (about) 100, (about) 150,(about) 200, (about) 250, (about) 300, (about) 350, (about) 400, (about)450, (about) 500, (about) 550, (about) 600, (about) 650, (about) 700,(about) 750, (about) 800, (about) 850, (about) 900, (about) 950, (about)1,000, (about) 1,100, (about) 1,200, (about) 1,300, (about) 1,400,(about) 1,500, (about) 1,600, (about) 1,700, (about) 1,800, (about)1,900, or (about) 2,000 amino acids in length, or of a range between anytwo of the foregoing. In some embodiments, at least 90% of the aminoacid residues of the XTEN are identified herein by glycine (G), alanine(A), serine (S), threonine (T), glutamate (E) or proline (P). In someembodiments, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or100% of the amino acid residues of the XTEN are glycine (G), alanine(A), serine (S), threonine (T), glutamate (E) or proline (P). In someembodiments, the XTEN comprises at least 4 different types of aminoacids are G, A, S, T, E, or P that is substantially randomized withrespect to any other nonoverlapping sequence motif comprising the XTENpolypeptide. In some embodiments, the XTEN (e.g., XTEN1, XTEN2, etc.) ischaracterized in that: (i) it comprises at least 100, or at least 150amino acids; (ii) at least 90% of the amino acid residues of the XTENare glycine (G), alanine (A), serine (S), threonine (T), glutamate (E)or proline (P); and (iii) it comprises at least 4 different types of theamino acids from G, A, S, T, E, or P that is substantially randomizedwith respect to any other nonoverlapping sequence motif comprising theXTEN polypeptide. One of ordinary skill in the art will understand that,as used herein, the term “glutamate” is a synonym for “glutamic acid,”and refers to the glutamic acid residue whether or not the side-chaincarboxyl is deprotonated. In some embodiments, the XTEN-containingfusion polypeptide comprises a first XTEN and a second XTEN. In someembodiments, the sum of the total number of amino acids in the firstXTEN and the total number of amino acids in the second XTEN is at least300, at least 350, at least 400, at least 500, at least 600, at least700, or at least 800 amino acids.

Non-Overlapping Sequence Motif

In some embodiments, the XTEN comprises, or is formed from, a pluralityof non-overlapping sequence motifs. In some embodiments, at least one ofthe non-overlapping sequence motifs is recurring (or repeated at leasttwo times in the XTEN), and wherein at least another one of thenon-overlapping sequence motifs is non-recurring (or found only oncewithin the XTEN). In some embodiments, the plurality of non-overlappingsequence motifs comprises (a) a set of (recurring) non-overlappingsequence motifs, wherein each non-overlapping sequence motif of the setof non-overlapping sequence motifs is repeated at least two times in theXTEN; and (b) a non-overlapping (non-recurring) sequence motif thatoccurs (or is found) only once within the XTEN. In some embodiments,each non-overlapping sequence motif is from 9 to 14 (or 10 to 14, or 11to 13) amino acids in length. In some embodiments, each non-overlappingsequence motif is 12 amino acids in length. In some embodiments, theplurality of non-overlapping sequence motifs comprises a set ofnon-overlapping (recurring) sequence motifs, wherein eachnon-overlapping sequence motif of the set of non-overlapping sequencemotifs is (1) repeated at least two times in the XTEN; and (2) isbetween 9 and 14 amino acids in length. In some embodiments, the set of(recurring) non-overlapping sequence motifs comprises 12-mer sequencemotifs identified herein by SEQ ID NOs: 179-200 and 1715-1722 inTable 1. In some embodiments, the set of (recurring) non-overlappingsequence motifs comprises 12-mer sequence motifs identified herein bySEQ ID NOs: 186-189 in Table 1. In some embodiments, the set of(recurring) non-overlapping sequence motifs comprise at least two, atleast three, or all four of 12-mer sequence motifs of SEQ ID NOs:186-189 in Table 1.

TABLE 1 Exemplary 12-Mer Sequence Motifs for Construction of XTENsMotif Family* Amino Acid Sequence SEQ ID NO. AD GESPGGSSGSES  182 ADGSEGSSGPGESS  183 AD GSSESGSSEGGP  184 AD GSGGEPSESGSS  185 AE, AMGSPAGSPTSTEE  186 AE, AM, AQ GSEPATSGSETP  187 AE, AM, AQ GTSESATPESGP 188 AE, AM, AQ GTSTEPSEGSAP  189 AF, AM GSTSESPSGTAP  190 AF, AMGTSTPESGSASP  191 AF, AM GTSPSGESSTAP  192 AF, AM GSTSSTAESPGP  193AG, AM GTPGSGTASSSP  194 AG, AM GSSTPSGATGSP  195 AG, AM GSSPSASTGTGP 196 AG, AM GASPGTSSTGSP  197 AQ GEPAGSPTSTSE  198 AQ GTGEPSSTPASE  199AQ GSGPSTESAPTE  200 AQ GSETPSGPSETA  179 AQ GPSETSTSEPGA  180 AQGSPSEPTEGTSA  181 BC GSGASEPTSTEP 1715 BC GSEPATSGTEPS 1716 BCGTSEPSTSEPGA 1717 BC GTSTEPSEPGSA 1718 BD GSTAGSETSTEA 1719 BDGSETATSGSETA 1720 BD GTSESATSESGA 1721 BD GTSTEASEGSAS 1722 *Denotesindividual motif sequences that, when used together in variouspermutations, results in a ″family sequence″

Barcode Fragment

In some embodiments, the polypeptide comprises a barcode fragment (e.g.,a first, second, or third barcode fragment of an XTEN) releasable fromthe polypeptide upon digestion by a protease. In some embodiments, thebarcode fragment (1) is a portion of the XTEN that includes at leastpart of the (non-recurring, non-overlapping) sequence motif that occurs(or is found) only once within the XTEN; and (2) differs in sequence andmolecular weight from all other peptide fragments that are releasablefrom the polypeptide upon complete digestion of the polypeptide by theprotease. One of ordinary skill in the art will understand that the term“barcode fragment” (or “barcode,” or “barcode sequence”) can refer toeither the portion of the XTEN cleavably fused within the polypeptide,or the resulting peptide fragment released from the polypeptide.

In some embodiments, the barcode fragment does not include theN-terminal amino acid or the C-terminal amino acid of the polypeptide.As described more fully below or described anywhere herein, in someembodiments, the barcode fragment is releasable (configured to bereleased) upon Glu-C digestion of the fusion polypeptide. In someembodiments, the barcode fragment does not include a glutamic acid thatis immediately adjacent to another glutamic acid, if present, in theXTEN. In some embodiments, the barcode fragment has a glutamic acid atits C-terminus. One of ordinary skill in the art will understand thatthe C-terminus of a barcode fragment can refer to the “last” (or themost C-terminal) amino acid residue within the barcode fragment, whencleavably fused within an XTEN, even if other “non-barcode” amino acidresidues are positioned C-terminal to the barcode fragment within thesame XTEN. In some embodiments, the barcode fragment has an N-terminalamino acid that is immediately preceded by a glutamic acid residue. Insome embodiments, the glutamic acid residue that precedes the N-terminalamino acid is not immediately adjacent to another glutamic acid residue.In some embodiments, the barcode fragment does not include a (second)glutamic acid residue at a position other than the C-terminus of thebarcode fragment unless the glutamic acid is immediately followed by aproline. In some embodiments, the barcode fragment is positioned adistance from either the N-terminus of the polypeptide or the C-terminusof the polypeptide, wherein the distance is from 10 to 150, or 10 to 125amino acids. In some embodiments, the barcode fragment is positionedwithin, or at a location of, 300, 280, 260, 250, 240, 220, 200, 190,180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 48, 40,36, 30, 24, 20, 12, or 10 amino acids from the N-terminus of thepolypeptide, or at a location in a range between any of the foregoing.In some embodiments, the barcode fragment is positioned within 200,within 150, within 100, or within 50 amino acids of the N-terminus ofthe polypeptide. In some embodiments, the barcode fragment is positionedat a location that is between 10 and 200, between 30 and 200, between 40and 150, or between 50 and 100 amino acids from the N-terminus of thepolypeptide. In some embodiments, the barcode fragment is positionedwithin, or at a location of, 300, 280, 260, 250, 240, 220, 200, 190,180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 48, 40,36, 30, 24, 20, 12, or 10 amino acids from the C-terminus of thepolypeptide, or at a location in a range between any of the foregoing.In some embodiments, the barcode fragment is positioned within 200,within 150, within 100, or within 50 amino acids of the C-terminus ofthe polypeptide. In some embodiments, the barcode fragment is positionedat a location that is between 10 and 200, between 30 and 200, between 40and 150, or between 50 and 100 amino acids from the C-terminus of thepolypeptide. In some embodiments, the barcode fragment (BAR) ischaracterized in that: (i) it does not include a glutamic acid that isimmediately adjacent to another glutamic acid, if present, in the XTEN;(ii) it has a glutamic acid at its C-terminus; (iii) it has anN-terminal amino acid that is immediately preceded by a glutamic acidresidue; and (iv) it is positioned a distance from either the N-terminusof the polypeptide or the C-terminus of the polypeptide, wherein thedistance is from 10 to 150 amino acids, or from 10 to 125 amino acids inlength. In some embodiments, the barcode fragment (i) does not includethe N-terminal amino acid or the C-terminal amino acid of thepolypeptide; (ii) does not include a glutamic acid that is immediatelyadjacent to another glutamic acid in the XTEN; (iii) has a glutamic acidat its C-terminus; (iv) has an N-terminal amino acid that is immediatelypreceded by a glutamic acid residue; and (v) is positioned a distancefrom either the N-terminus of the polypeptide or the C-terminus of thepolypeptide, wherein the distance is from 10 to 150, or 10 to 125 aminoacids in length. In some embodiments, the glutamic acid residue thatprecedes the N-terminal amino acid is not immediately adjacent toanother glutamic acid residue. In some embodiments, the barcode fragmentdoes not include a glutamic acid residue at a position other than theC-terminus of the barcode fragment unless the glutamic acid isimmediately followed by a proline. One or ordinary skill in the art willunderstand the term “distance,” as used herein, can refer to the numberof amino acid residues from the N-terminus of the polypeptide to themost N-terminal amino acid residue of the barcode fragment, or from theC-terminus of the polypeptide to the most C-terminal amino acid residueof the barcode fragment. In some embodiments, for a barcoded XTEN fusedto a biologically-active polypeptide, at least one barcode fragment (orat least two barcode fragments, or three barcode fragments) contained inthe barcoded XTEN is positioned at least 50, 75, 100, 125, 150, 175,200, 225, 250, 275, 300 amino acids from the biologically activepolypeptide. In some embodiments, the barcode fragment is at least 4, atleast 5, at least 6, at least 7, or at least 8 amino acids in length. Insome embodiments, the barcode fragment is at least 4 amino acids inlength. In some embodiments, the barcode fragment is 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 aminoacids in length, or in a range between any of the foregoing values. Insome embodiments, the barcode fragment is between 4 and 20, between 5and 15, between 6 and 12, or between 7 and 10 amino acids in length. Insome embodiments, the barcode fragment comprises an amino acid sequenceidentified herein by SEQ ID NOs: 68-77 in Table 2.

TABLE 2 Exemplary Barcode Fragments Releasable Upon  Glu-C DigestAmino Acid Sequence SEQ ID NO: SPATSGSTPE 68 (BAR001) GSAPATSE69 (BAR002) GSAPGTATE 70 (BAR003) GSAPGTE 71 (BAR004) PATSGPTE72 (BAR005) SASPE 73 (BAR006) PATSGSTE 74 (BAR007) GSAPGTSAE 75 (BAR008)SATSGSE 76 (BAR009) SGPGSTPAE 77 (BAR010)

In some embodiments of the polypeptides of this disclosure, the XTEN canhave a length defined by a proximal end and a distal end, where (1) theproximal end is positioned, relative to the distal end, closer to thebispecific antibody construct (BsAb), and where (2) the barcode fragment(BAR) can be positioned within a region of the XTEN that extends, asmeasured from the distal end, between 5% and 50%, between 7% and 40%, orbetween 10% and 30% of the length of the XTEN.

In some embodiments of the polypeptides of this disclosure, the XTENfurther comprises additional one or more barcode fragments, wherein saidadditional one or more barcode fragments each differ in sequence andmolecular weight from all other peptides fragments that are releasablefrom said polypeptide upon complete digestion of said polypeptide bysaid protease. In some embodiments, a barcoded XTEN comprises only onebarcode fragment. In some embodiments, a barcoded XTEN comprises a setof barcode fragments, comprising a first barcode fragment, such as thosedescribed above or anywhere else herein. In some embodiments, the set ofbarcode fragments comprises a second barcode fragment (or a furtherbarcode fragment), such as those described above or anywhere elseherein. In some embodiments, the set of barcode fragments comprises athird barcode fragment, such as those described above or anywhere elseherein. The set of barcode fragments fused within an N-terminal XTEN canbe referred to as an N-terminal set of barcodes (“an N-terminal set”).The set of barcode fragments fused within a C-terminal XTEN can bereferred to as a C-terminal set of barcodes (“a C-terminal set”). Insome embodiments, the N-terminal set comprises a first barcode fragmentand a second barcode fragment. In some embodiments, the N-terminal setfurther comprises a third barcode fragment. In some embodiments, theC-terminal set comprises a first barcode fragment and a second barcodefragment. In some embodiments, the C-terminal set further comprises athird barcode fragment. In some embodiments, the second barcode fragmentis positioned N-terminal to the first barcode fragment of the same set.In some embodiments, the second barcode fragment is positionedC-terminal to the first barcode fragment of the same set. In someembodiments, the third barcode fragment is positioned N-terminal to boththe first and second barcode fragments. In some embodiments, the thirdbarcode fragment is positioned C-terminal to both the first and secondbarcode fragments. In some embodiments, the third barcode fragment ispositioned between the first and second barcode fragments. In someembodiments, the polypeptide comprises a set of barcode fragments thatincludes a first barcode fragment, a further (second) barcode fragment,and at least one additional barcode fragment, wherein each barcodefragment of the set of barcode fragments (1) is a portion of the secondXTEN and (2) differs in sequence and molecular weight from all otherpeptides fragments that are releasable from the polypeptide uponcomplete digestion of the polypeptide by the protease.

Exemplary Barcoded XTEN

Amino acid sequences of 13 exemplary barcoded XTENs, containing onebarcode (e.g., SEQ ID NOs: 8002-8003, 8005-8009, and 8013), or twobarcodes (e.g., SEQ ID NOS: 8001, 8004, and 8012), or three barcodes(e.g., SEQ ID NO: 8011), are illustrated in Table 3a. Among these 13exemplary barcoded XTEN, six (SEQ ID NOs: 8001-8003, 8008-8009, and8011) are to be fused to a biologically-active protein at the C-terminalof the biologically-active protein, and seven (SEQ ID NOS: 8004-8007,8010, and 8012-8013) are to be fused at the N-terminal of thebiologically-active protein. In some embodiments, the XTEN has at least90%, at least 92%, at least 95%, at least 98%, at least 99% or 100%sequence identity to a sequence identified herein by SEQ ID NOs:8001-8020 in Table 3a.

TABLE 3a Exemplary Barcoded XTENs SEQ ID XTEN # of Total # NO. TypeBarcode(s) Amino Acid Sequence of AAs 8001 C-terminal 2PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGS  864 XTENPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGftabTSESATPESGPGS E PATSGPTESGSEPATSGSETPGSPAGSPTSTEEGTSTEPSE GSAPGTESTPSEGSAPGSEPATSGSETPGTSESATPESGPGT STEPSEGSAPGEPEA 8002 C-terminal 1PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGS  864 XTENPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSE PA TSGPTESGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPS EGSAPGEPEA 8003 C-terminal 1PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGS  864 XTENPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSE GSAP GTESTPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPS EGSAPGEPEA 8004 N-terminal 2ASSPAGSPTSTESGTSESATPESGPGTETEPSEGSAPGTSESA  288 XTENTPESGPGSEPATSGSETPGTSESATPE SGPGSTPAE SGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESAT PESGPGE SPATSGSTPEGTSESATPESGPGSPAGSPTSTEEGS PAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGGSAP 8005 N-terminal 1ASSPAGSPTSTESGTSESATPESGPGTSTEPSEGSAPGTSESA  288 XTENTPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATP ESGPGE SPATSGSTPEGTSESATPESGPGSPAGSPTSTEEGSP AGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGGSAP 8006 N-terminal 1ASSPAGSPTSTESGTSESATPESGPGTSTEPSEGSAPGTSESA  288 XTENTPESGPGSEPATSGSETPGTSESATPE SGPGSTPAE SGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGEEPATSGSTPEGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGGSAP 8007 N-terminal 1ASSPAGSPTSTESGTSESATPESGPGTSTEPSEGSAPGTSESA  288 XTENTPESGPGSEPATSGSETPGTSESATPE SGPGSTPAE SGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGGSAP 8008 C-terminal 1PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGS  864 XTENPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSE GSAP GTESTPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPS EGSAPG 8009 C-terminal 1PGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESA  576TPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTE EGTSTEPSE GSAPGTESTPSEGSAPGSEPATSGSETPGTSES ATPESGPGTSTEPSEGSAPG 8010 XTEN 2SAGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGS 1152 N-terminalPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPE XTEN SGPGSTPAESGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTE PSEGSAPGTSESATPESGPGSEPATSGSTE TPGTSTEPSEGSA PGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTESAS 8011 C-terminal 3SAGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGS 1152 XTENPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSES ATPESGPGSEPATSGSETPGSEPATSGSTE TPGSPAGSPTSTE EGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSE GSAPGTATE SPEGSAPGTSESATPESGP GTSTEPSE GSAPGTSAESATPESGPGSEPATSGSETPGTSTE PSEGSAPGTSTEPSEGSAPGTSESATPESGPGTESAS 8012N-terminal 2 GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSP  864 XTENTSTEEGTSTEPSEGSAPGTSTEPSE GSAPATSE SATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSE SASPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSE GSAP 8013 N-terminal 1GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSP  864 XTENTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGS E SATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSE GSAP 8014 N-terminal 1SPAGSPTSTESGTSESATPESGPGTSTEPSEGSAPGTSESATP  292 XTENESGPGSEPATSGSETPGTSESATPE SGPGSTPAESGSE TPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGGSAP 8015 C-terminal 1PGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESA  582 XTENTPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTE EGTSTEPSE GSAPGTESTPSEGSAPGSEPATSGSETPGTSES ATPESGPGTSTEPSEGSAPGEPEA 8016 C-terminal 1TPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPG  576 XTENSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSE SAT SGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPG SEPATSGSETPGTSESA 8017C-terminal 1 GTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATS  576 XTENGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPES GPGTSTEPSEGSAPGTSE SASPESGPGSPAGSPTSTEEGSPAG SPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATS GSETPGTSESATPESGP 8018C-terminal 1 GSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGT  576 XTENSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAP GTSESATPESGPGSE PATSGSTETGTSESATPESGPGSEPAT SGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATS 8019 C-terminal 1EGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGT  576 XTENSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSES ATPESGPGSEPATSGSETPGTSESASPE SGPGTSTEPSEGSAP GSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGT SESATPESGPGTSESAT 8020N-terminal 1 ASSPAGSPTSTESGTSESATPESGPGTSTEPSEGSAPGTSESA  294 XTENTPESGPGSEPATSGSETPGTSESATPE SGPGSTPA ESGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGGSAP

In some embodiments, a barcoded XTEN can be obtained by making one ormore mutations to a general-purpose XTEN, such as any listed in Table3b, according to one or more of the following criteria: to minimize thesequence change in the XTEN, to minimize the amino acid compositionchange in the XTEN, to substantially maintain the net charge of theXTEN, to substantially maintain (or improve) low immunogenicity of theXTEN, and to substantially maintain (or improve) the pharmacokineticproperties of the XTEN. In some embodiments, the XTEN sequence has atleast 90%, at least 92%, at least 95%, at least 98%, at least 99%, or10000 sequence identity to any one of SEQ ID NOs: 601-659 listed inTable 3b. In some embodiments, the XTEN sequence, having at least 90%(e.g., at least 92%, at least 95%, at least 98%, or at least 99%) butless than 100% sequence identity to any of SEQ ID NOs: 601-659 listed inTable 3b, is obtained by one or more mutations (e.g., less than 10, lessthan 8, less than 6, less than 5, less than 4, less than 3, less than 2mutations) of the corresponding sequence from Table 3b. In someembodiments, the one or more mutations comprise deletion of a glutamicacid residue, insertion of a glutamic acid residue, substitution of aglutamic acid residue, or substitution for a glutamic acid residue, orany combination thereof. In some embodiments, where the XTEN sequencediffers from, but has at least 90% (e.g., at least 92%, at least 95%, atleast 98%, or at least 99%) sequence identity to, any one of SEQ ID NOs:601-659 listed in Table 3b, at least 80%, at least 90%, at least 95%, atleast 97%, or about 100% of the difference between the XTEN sequence andthe corresponding sequence of Table 3b involve deletion of a glutamicacid residue, insertion of a glutamic acid residue, substitution of aglutamic acid residue, or substitution for a glutamic acid residue, orany combination thereof. In some such embodiments, at least 80%, atleast 90%, at least 95%, at least 97%, or about 100% of the differencebetween the XTEN sequence and the corresponding sequence of Table 3binvolve a substitution of a glutamic acid residue, or a substitution fora glutamic acid residue, or both. The term “a substitution of a firstamino acid,” as used herein, refers to replacement of the first aminoacid residue for a second amino acid residue, resulting in the secondamino acid residue taking place at the substitution position in theobtained sequence. For example, “a substitution of glutamic acid” refersto replacement of the glutamic acid (E) residue for a non-glutamic acidresidue (e.g., serine (S)). The term “a substitution for a first aminoacid,” as used herein, refers to replacement of a second amino acidresidue for the first amino acid residue, resulting in the first aminoacid residue taking place at the substitution position in the obtainedsequence. For example, “a substitution for glutamic acid” refers toreplacement of a non-glutamic acid residue (e.g., serine (S)) for aglutamic acid residue.

TABLE 3bExemplary General-Purpose XTEN for Engineering into Barcoded XTEN(s)XTEN SEQ ID Name Amino Acid Sequence NO AE144GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGS 601APGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAP AE144_1ASPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAP 602GTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPG AE144_2ATSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGP 603GTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPG AE144_2BTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGP 604GTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPG AE144_3ASPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP 605GTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPG AE144_3BSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP 606GTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPG AE144_4ATSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGP 607GTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPG AE144_4BTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGP 608GTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPG AE144_5ATSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGP 609GTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEG AE144_6BTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETP 610GSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPG AE288_1GTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPES 611GPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP AE288_2GSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGS 612APGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAP AE576GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGS 613APGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAP AE624MAEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGSPAGSPTS 614TEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAP AE864GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGS 615APGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP AE865GGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEG 616SAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP AE866PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGS 617APGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPG AE1152GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGS 618APGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP AE144ASTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETP 619GTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGS AE144BSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP 620GSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPG AE180ATSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAG 621SPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGS EPATS AE216APESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSES 622ATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESAT AE252AESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESA 623TPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSE AE288ATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEP 624ATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESA AE324APESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEP 625SEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATS AE360APESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAG 626SPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSG SETPGTSESATAE396A PESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAG 627SPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSE GSAPGTSTEPSAE432A EGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSES 628ATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATS AE468AEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSES 629ATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSES AT AE504AEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAG 630SPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPS AE540ATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTST 631EPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEP AE576ATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSES 632ATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESA AE612AGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAG 633SPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESAT AE648APESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEP 634SEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETP GTSESATAE684A EGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTE 635PSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATS AE720ATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTS 636TEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTE AE756ATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTS 637TEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSES AE792AEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSES 638ATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPS AE828APESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSES 639ATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESAT AE869GSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSE 640GSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGR AE144_R1SAGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTE 641PSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTESASR AE288_R1SAGSPTGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGT 642STEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPSASR AE432_R1SAGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTE 643PSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTESASR AE576_R1SAGSPTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGT 644STEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPSASR AE864_R1SAGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTE 645PSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTESASR AE712PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGS 646APGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGS PAGSPTSTEAHHHAE864_R2 GSPGAGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTE 647PSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTESASR AE288_3SPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETP 648GTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPG AE284GTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPES 649GPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSE AE292SPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETP 650GTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGGSA P AE864_2AGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPG 651TSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGAAEPEA AE867GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGS 652APGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGAAEPEA AE867_2SPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEG 653SAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPG AE868PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGS 654APGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGAAEPEA AE144_7AGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGS 655APGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAP AE292SPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETP 656GTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGGSA P AE293PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGS 657APGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPEGAAEPE A AE300PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGS 658APGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSP AGAAEPEAAE584 PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGS 659APGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGAAEPE A

In some embodiments, for constructing the sequence of a barcoded XTEN,amino-acid mutations are performed on XTEN of intermediate lengths tothose of Table 3b, as well as XTEN of longer lengths than those of Table3b, such as those in which one or more 12-mer motifs of Table 1 areadded to the N- or C-terminus of a general-purpose XTEN of Table 3b.

Additional examples of general-purpose XTEN sequences that can be usedaccording to the present disclosure are disclosed in U.S. PatentPublication Nos. 2010/0239554 A1, 2010/0323956 A1, 2011/0046060 A1,2011/0046061 A1, 2011/0077199 A1, or 2011/0172146 A1, or InternationalPatent Publication Nos. WO 2010091122 A1, WO 2010144502 A2, WO2010144508 A1, WO 2011028228 A1, WO 2011028229 A1, WO 2011028344 A2, WO2014/011819 A2, or WO 2015/023891.

In some embodiments, a barcoded XTEN fused within a polypeptide chainadjacent to the N-terminus of the polypeptide chain (“N-terminal XTEN”)can be attached to a His tag of HHHHHH (SEQ ID NO: 48) or HHHHHHHH (SEQID NO: 49) at the N-terminus to facilitate the purification of thefusion polypeptide. In some embodiments, a barcoded XTEN fused within apolypeptide chain at the C-terminus of the polypeptide chain(“C-terminal XTEN”) can be comprise or be attached to the sequence EPEAat the C-terminus to facilitate the purification of the fusionpolypeptide. In some embodiment, the fusion polypeptide comprises bothan N-terminal barcoded XTEN and a C-terminal barcoded XTEN, wherein theN-terminal barcoded XTEN is attached to a His tag of HHHHHH (SEQ ID NO:48) or HHHHHHHH (SEQ ID NO: 49) at the N-terminus; and wherein theC-terminal barcoded XTEN is attached to the sequence EPEA at theC-terminus, thereby facilitating purification of the fusion polypeptide,for example, to at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, orat least 99% purity by chromatography methods known in the art,including but not limited to IMAC chromatography, C-tagXL affinitymatrix, and other such methods, including but not limited to thosedescribed in the EXAMPLES section below.

Protease Digestion

A barcode fragment, as described above or anywhere else herein, can becleavably fused within the XTEN and releasable (configured to bereleased) from the XTEN upon digestion of the polypeptide by a protease.In some embodiments, the protease is a Glu-C protease. In someembodiments, the protease cleaves on the C-terminal side of glutamicacid residues that are not followed by proline. One of ordinary skill inthe art will understand that a barcoded XTEN (an XTEN that containsbarcode fragment(s) therewithin) is designed to achieve high efficiency,precision and accuracy of the protease digestion. For example, one ofordinary skill in the art will understand that adjacent Glu-Glu (EE)residues in an XTEN sequence can result in varying cleavage patternsupon Glu-C digestion. Accordingly, when Glu-C protease is used forbarcode release, the barcoded XTEN or the barcode fragment(s) may notcontain any Glu-Glu (EE) sequence. One of ordinary skill in the art willalso understand that a di-peptide Glu-Pro (EP) sequence, if present inthe fusion polypeptide, may not be cleaved by Glu-C protease during thebarcode release process.

Structural Configuration of BPXTEN

In some embodiments, a BPXTEN fusion protein comprises a single BP and asingle XTEN. Such BPXTEN can have at least the following permutations ofconfigurations, each listed in an N- to C-terminus orientation: BP-XTEN;XTEN-BP; BP-S-XTEN; and XTEN-S-BP.

In some embodiments, the BPXTEN comprises a C-terminal XTEN and,optionally, a spacer sequence (S) (such as one described herein, e.g.,in Table C) between the XTEN and the BP. Such BPXTEN can be representedby Formula I (depicted N- to C-terminus):

(BP)-(S)_(x)-(XTEN)  (I),

wherein BP is a biologically active protein as described hereinbelow; Sis a spacer sequence (such as one described herein, e.g., in Table C)having between 1 to about 50 amino acid residues that can optionallyinclude a BP release segment (as described more fully hereinbelow); x iseither 0 or 1; and XTEN can be any one as described hereinabove oranywhere else herein.

In some embodiments, the BPXTEN comprises an N-terminal XTEN and,optionally, a spacer sequence (S) (such as one described herein, e.g.,in Table C) between the XTEN and the BP. Such BPXTEN can be representedby Formula II (depicted N- to C-terminus):

(XTEN)-(S)_(x)-(BP)  (II),

wherein BP is a biologically active protein as described hereinbelow; Sis a spacer sequence (such as one described herein, e.g., in Table C)having between 1 to about 50 amino acid residues that can optionallyinclude a BP release segment (as described more fully hereinbelow); x iseither 0 or 1; and XTEN can be any one as described hereinabove oranywhere else herein.

In some embodiment, the BPXTEN comprises both an N-terminal XTEN and aC-terminal XTEN. Such BPXTEN (e.g., the XPATs in FIGS. 1-2 ) can berepresented by Formula III:

(III) (XTEN)-(S)_(y)-(BP)-(S)_(z)-(XTEN)wherein BP is a biologically active protein as described hereinbelow; Sis a spacer sequence (such as one described herein, e.g., in Table C)having between 1 to about 50 amino acid residues that can optionallyinclude a BP release segment (as described more fully hereinbelow); y iseither 0 or 1; z is either 0 or 1; and XTEN can be any one as describedhereinabove or anywhere else herein.

Biologically Active Polypeptide

A biologically active protein (BP) fused to XTEN (as described hereinabove or described anywhere else herein), particularly those disclosedhereinbelow, comprising sequences identified herein by those of Tables6a-6f, together with their corresponding nucleic acid and amino acidsequences, are well known in the art. Descriptions and sequences ofthese BP are available in public databases such as Chemical AbstractsServices Databases (e.g., the CAS Registry), GenBank, The UniversalProtein Resource (UniProt) and subscription provided databases such asGenSeq (e.g., Derwent). Polynucleotide sequences may be a wild typepolynucleotide sequence encoding a given BP (e.g., either full length ormature), or in some instances the sequence may be a variant of the wildtype polynucleotide sequence (e.g., a polynucleotide which encodes thewild type biologically active protein), wherein the DNA sequence of thepolynucleotide has been optimized, for example, for expression in aparticular species; or a polynucleotide encoding a variant of the wildtype protein, such as a site directed mutant or an allelic variant. Itis well within the ability of the skilled artisan to use a wild-type orconsensus cDNA sequence or a codon-optimized variant of a BP to createBPXTEN constructs contemplated by the invention using methods known inthe art and/or in conjunction with the guidance and methods providedherein.

The BP for inclusion in the BPXTEN (a fusion polypeptide comprising atleast one BP and at least one XTEN) can include any protein of biologic,therapeutic, prophylactic, or diagnostic interest or function, or thatis useful for mediating a biological activity or preventing orameliorating a disease, disorder or conditions when administered to asubject. Of particular interest are BP for which an increase in apharmacokinetic parameter, increased solubility, increased stability,masking of activity, or some other enhanced pharmaceutical property issought, or those BP for which increasing the terminal half-life wouldimprove efficacy, safety, or result in reduce dosing frequency and/orimprove patient compliance. Thus, the BPXTEN fusion protein compositionsare prepared with various objectives in mind, including improving thetherapeutic efficacy of the bioactive compound by, for example,increasing the in vivo exposure or the length that the BPXTEN remainswithin the therapeutic window when administered to a subject, comparedto a BP not linked to XTEN.

A BP can be a native, full-length protein or can be a fragment or asequence variant of a biologically active protein that retains at leasta portion of the biological activity of the native protein.

In one embodiment, the BP incorporated into the subject compositions canbe a recombinant polypeptide with a sequence corresponding to a proteinfound in nature. In some embodiments, the BP can be sequence variants,fragments, homologs, and mimetics of a natural sequence that retain atleast a portion of the biological activity of the native BP. Innon-limiting examples, a BP can be a sequence that exhibits at leastabout 80% sequence identity, or alternatively 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% sequence identity to a protein sequence identified herein. Infurther non-limiting examples, a BP can be a bispecific sequencecomprising a first binding domain and a second binding domain, whereinthe first binding domain, having specific binding affinity to atumor-specific marker or an antigen of a target cell, exhibits at leastabout 80% sequence identity, or alternatively 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% sequence identity to paired VL and VH sequences of an anti-CD3antibody identified herein by Table 6f; and wherein the second bindingdomain, having specific binding affinity to an effector cell, exhibitsat least about 80% sequence identity, or alternatively 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% sequence identity to paired VL and VH sequences of ananti-target cell antibody identified herein by Table 6a. In oneembodiment, a BPXTEN fusion protein can comprise a single BP moleculelinked to an XTEN. In some embodiments, the BPXTEN can comprise a firstBP and a second molecule of the same BP, resulting in a fusion proteincomprising the two BP linked to one or more XTEN (for example, twomolecules of glucagon, or two molecules of hGH).

In general, BP will exhibit a binding specificity to a given target (ora given number of targets) or another desired biological characteristicwhen used in vivo or when utilized in an in vitro assay. For example,the BP can be an agonist, a receptor, a ligand, an antagonist, anenzyme, an antibody (e.g., mono- or bi-specific), or a hormone. Ofparticular interest are BP used or known to be useful for a disease ordisorder wherein the native BP have a relatively short terminalhalf-life and for which an enhancement of a pharmacokinetic parameter(which optionally could be released from the fusion protein by cleavageof a spacer sequence) would permit less frequent dosing or an enhancedpharmacologic effect. Also of interest are BP that have a narrowtherapeutic window between the minimum effective dose or bloodconcentration (Cmin) and the maximum tolerated dose or bloodconcentration (Cmax). In such cases, the linking of the BP to a fusionprotein comprising a select XTEN sequence(s) can result in animprovement in these properties, making them more useful as therapeuticor preventive agents compared to BP not linked to XTEN.

The BP encompassed by the inventive compositions can have utility in thetreatment in various therapeutic or disease categories, including butnot limited to glucose and insulin disorders, metabolic disorders,cardiovascular diseases, coagulation and bleeding disorders, growthdisorders or conditions, endocrine disorders, eye diseases, kidneydiseases, liver diseases, tumorigenic conditions, inflammatoryconditions, autoimmune conditions, etc.

“Anti-CD3” means the monoclonal antibody against the T cell surfaceprotein CD3, species and sequence variants, and fragments thereof,including OKT3 (also called muromonab) and humanized anti-CD3 monoclonalantibody (hOKT31(Ala-Ala))(KC Herold et al., New England Journal ofMedicine 346:1692-1698. 2002) Anti-CD3 prevents T-cell activation andproliferation by binding the T-cell receptor complex present on alldifferentiated T cells. Anti-CD3-containing fusion proteins of theinvention may find particular use to slow new-onset Type 1 diabetes,including use of the anti-CD3 as a therapeutic effector as well as atargeting moiety for a second therapeutic BP in the BPXTEN composition.The sequences for the variable region and the creation of anti-CD3 havebeen described in U.S. Pat. Nos. 5,885,573 and 6,491,916.

The BP of the subject compositions are not limited to native,full-length polypeptides, but also include recombinant versions as wellas biologically and/or pharmacologically active variants or fragmentsthereof. For example, it will be appreciated that various amino acidsubstitutions can be made in the GP to create variants without departingfrom the spirit of the invention with respect to the biological activityor pharmacologic properties of the BP. Examples of conservativesubstitutions for amino acids in polypeptide sequences are shown inTable 5. However, in embodiments of the BPXTEN in which the sequenceidentity of the BP is less than 100% compared to a specific sequencedisclosed herein, the invention contemplates substitution of any of theother 19 natural L-amino acids for a given amino acid residue of thegiven BP, which may be at any position within the sequence of the BP,including adjacent amino acid residues. If any one substitution resultsin an undesirable change in biological activity, then one of thealternative amino acids can be employed and the construct evaluated bythe methods described herein, or using any of the techniques andguidelines for conservative and non-conservative mutations set forth,for instance, in U.S. Pat. No. 5,364,934, the contents of which isincorporated by reference in its entirety, or using methods generallyknown to those of skill in the art. In addition, variants can alsoinclude, for instance, polypeptides wherein one or more amino acidresidues are added or deleted at the N- or C-terminus of the full-lengthnative amino acid sequence of a BP that retains at least a portion ofthe biological activity of the native peptide.

TABLE 5 Exemplary conservative amino acid substitutions Original ResidueExemplary Substitutions Ala (A) val; leu; ile Arg (R) lys; gin; asn Asn(N) gin; his; lys; arg Asp (D) glu Cys (C) ser Gln (Q) asn Glu (E) aspGly (G) pro His (H) asn: gin: Iys: arg xIle (I) leu; val; met; ala; phe:norleucine Leu (L) norleucine: ile: val; met; ala: phe Lys (K) arg: gin:asn Met (M) leu; phe; ile Phe (F) leu: val: ile; ala Pro (P) gly Ser (S)thr Thr (T) ser Trp (W) tyr Tyr(Y) trp: phe: thr: ser Val (V) ile; leu;met; phe; ala; norleucine

In some embodiments, a BP incorporated into a BPXTEN fusion protein canhave a sequence that exhibits at least about 80% sequence identity to asequence, alternatively at least about 81%, or about 82%, or about 83%,or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, orabout 89%, or about 90%, or about 91%, or about 92%, or about 93%, orabout 94%, or about 95%, or about 96%, or about 97%, or about 98%, orabout 99%, or 100% sequence identity. In some embodiments, a BPincorporated into a BPXTEN can be a bispecific sequence comprising afirst binding domain and a second binding domain, wherein the firstbinding domain, having specific binding affinity to a tumor-specificmarker or an antigen of a target cell, exhibits at least about 80%sequence identity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to paired VL and VH sequences of an anti-CD3 antibodyidentified herein by Table 6f; and wherein the second binding domain,having specific binding affinity to an effector cell, exhibits at leastabout 80% sequence identity, or alternatively 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% sequence identity to paired VL and VH sequences of an anti-targetcell antibody identified herein by Table 6a. The BP of the foregoingembodiments can be evaluated for activity using assays or measured ordetermined parameters as described herein, and those sequences thatretain at least about 40%, or about 50%, or about 55%, or about 60%, orabout 70%, or about 80%, or about 90%, or about 95% or more activitycompared to the corresponding native BP sequence would be consideredsuitable for inclusion in the subject BPXTEN. The BP found to retain asuitable level of activity can be linked to one or more XTENpolypeptides described hereinabove or anywhere else herein. In oneembodiment, a BP found to retain a suitable level of activity can belinked to one or more XTEN polypeptides, having at least about 80%sequence identity (e.g., at least about 81%, at least about 82%, atleast about 83%, at least about 84%, at least about 85%, at least about86%, at least about 87%, at least about 88%, at least about 89%, atleast about 90%, at least about 91%, at least about 92%, at least about93%, at least about 94%, at least about 95%, at least about 96%, atleast about 97%, at least about 98%, at least about 99%, or 100%sequence identity) to a sequence from Tables 3a-3b, resulting in achimeric fusion protein.

T Cell Engagers

Additional structural configuration formulae of BPXTEN relate toXTENylated Protease-Activated T Cell Engagers (“XPAT” or “XPATs”),wherein BP is a bispecific antibody (e.g., a bispecific T-cell engager).In some embodiments, the XPAT composition comprises (1) a first portioncomprising a first binding domain and a second binding domain, and (2) asecond portion comprising the release segment, and (3) a third portioncomprising the bulking moiety. In some embodiments, the XPAT compositionhas the configuration of Formula Ia (depicted N-terminus to C-terminus):

(first portion)-(second portion)-(third portion)  (Ia)

wherein first portion is a bispecific comprising two scFv wherein thefirst binding domain has specific binding affinity to a tumor-specificmarker or an antigen of a target cell and the second binding domain hasspecific binding affinity to an effector cell; the second portioncomprises a release segment (RS) capable of being cleaved by a mammalianprotease (as described more fully hereinbelow, the protease can betumor- or antigen-specific, thereby activation); and the third portionis a bulking moiety. In the foregoing embodiment, the first portionbinding domains can be in the order (VL-VH)1-(VL-VH)2, wherein “1” and“2” represent the first and second binding domains, respectively, or(VL-VH)1-(VH-VL)2, or (VH-VL)1-(VL-VH)2, or (VH-VL)1-(VH-VL)2, whereinthe paired binding domains are linked by a polypeptide linker (asdescribed more fully hereinbelow). In one embodiment, the first portionVL and VH are set forth in Tables 6a-6f; RS is identified herein by thegroup of sequences set forth in Tables 8a-8b (as described more fullyhereinbelow); and the bulking moiety is XTEN; albumin binding domain;albumin; IgG binding domain; polypeptides consisting of proline, serine,and alanine; fatty acid; Fc domain; polyethylene glycol (PEG), PLGA; orhydoxylethyl starch. Where desired, the bulking moiety is an XTEN havingat least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to a sequence comprising the group of sequences setforth in Tables 3a-3b. In the foregoing embodiments, the composition isa recombinant fusion protein. In some embodiments, the portions arelinked by chemical conjugation.

In some embodiments, the XPAT composition has the configuration ofFormula IIa (depicted N-terminus to C-terminus):

(third portion)-(second portion)-(first portion)  (IIa)

wherein first portion is a bispecific comprising two scFv wherein thefirst binding domain has specific binding affinity to a tumor-specificmarker or an antigen of a target cell and the second binding domain hasspecific binding affinity to an effector cell; the second portioncomprises a release segment (RS) capable of being cleaved by a mammalianprotease; and the third portion is a bulking moiety. In the foregoingembodiment, the first portion binding domains can be in the order(VL-VH)1-(VL-VH)2, wherein “1” and “2” represent the first and secondbinding domains, respectively, or (VL-VH)1-(VH-VL)2, or(VH-VL)1-(VL-VH)2, or (VH-VL)1-(VH-VL)2, wherein the paired bindingdomains are linked by a polypeptide linker as described herein, below.In one embodiment, the first portion VL and VH are identified in Tables6a-6f; RS is identified herein as the group of sequences set forth inTables 8a-8b; and the bulking moiety is XTEN; albumin binding domain;albumin; IgG binding domain; polypeptides consisting of proline, serine,and alanine; fatty acid; or Fc domain. Where desired, the bulking moietyis an XTEN having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% sequence identity to a sequence comprising the groupof sequences set forth in Tables 3a-3b. In the foregoing embodiments,the composition is a recombinant fusion protein. In some embodiments,the portions are linked by chemical conjugation.

In some embodiments, the XPAT composition has the configuration ofFormula IIIa (depicted N-terminus to C-terminus):

(fifth portion)-(fourth portion)-(first portion)-(second portion)-(thirdportion)  (IIIa)

wherein first portion is a bispecific comprising two scFv wherein thefirst binding domain has specific binding affinity to a tumor-specificmarker or an antigen of a target cell and the second binding domain hasspecific binding affinity to an effector cell; the second portioncomprises a release segment (RS) capable of being cleaved by a mammalianprotease; the third portion is a bulking moiety; the fourth portioncomprises a release segment (RS) capable of being cleaved by a mammalianprotease which may be identical or different from the second portion;and the fifth portion is a bulking moiety that may be identical or maybe different from the third portion. In the foregoing embodiment, thefirst portion binding domains can be in the order (VL-VH)1-(VL-VH)2,wherein “1” and “2” represent the first and second binding domains,respectively, or (VL-VH)1-(VH-VL)2, or (VH-VL)1-(VL-VH)2, or(VH-VL)1-(VH-VL)2, wherein the paired binding domains are linked by apolypeptide linker as described herein, below. In the foregoingembodiments, the RS is identified as sequences set forth in Tables8a-8b. In the foregoing embodiments, the bulking moiety is XTEN; albuminbinding domain; albumin; IgG binding domain; polypeptides consisting ofproline, serine, and alanine; fatty acid; or Fc domain. Where desired,the bulking moiety is an XTEN having at least about 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequenceidentified herein by the sequences set forth in Tables 3a-3b. In theforegoing embodiments, the composition is a recombinant fusion protein.In some embodiments, the portions are linked by chemical conjugation.

The subject compositions, based on their design and specific components,advantageously provide bispecific therapeutics that have moreselectivity, greater half-life, and result in less toxicity and fewerside effects once they are cleaved by proteases found in associated withthe target tissues or tissues rendered unhealthy by a disease, such thatthe subject compositions have improved therapeutic index compared tobispecific antibody compositions known in the art. Such compositions areuseful in the treatment of certain diseases, including, but not limitedto cancer. It will be appreciated by those of skill in the art that thecompositions of the instant invention achieve this reduction innon-specific interactions by a combination of mechanism, which includesteric hindrance by locating the binding domains to the bulky XTENmolecules, steric hindrance in that the flexible, unstructuredcharacteristic of the long flexible XTEN polypeptides, by being tetheredto the composition, are able to oscillate and move around the bindingdomains, providing blocking between the composition and tissues orcells, as well as a reduction in the ability of the intact compositionto penetrate a cell or tissue due to the large molecular mass(contributed to by both the actual molecular weight of the XTEN and dueto the large hydrodynamic radius of the unstructured XTEN) compared tothe size of the individual binding domains. However, the compositionsare designed such that when in proximity to a target tissue or cellbearing or secreting a protease capable of cleaving the RS, or wheninternalized into a target cell or tissue when a binding domain hasbound the ligand, the bispecific binding domains are liberated from thebulk of the XTEN by the action of the protease(s), removing the sterichindrance barrier, and is freer to exert its pharmacologic effect. Thesubject compositions find use in the treatment of a variety ofconditions where selective delivery of a therapeutic bispecific antibodycomposition to a cell, tissue or organ is desired. In one embodiment,the target tissue is a cancer, which may be a leukemia, a lymphoma, or atumor of an organ or system.

Binding Domains

The disclosure contemplates use of single chain binding domains, such asbut not limited to Fv, Fab, Fab′, Fab′-SH, F(ab′)2, linear antibodies,single domain antibody, single domain camelid antibody, single-chainantibody molecules (scFv), and diabodies capable of binding ligands orreceptors associated with effector cells and antigens of diseasedtissues or cells that are cancers, tumors, or other malignant tissues.In some embodiments, an antigen binding fragment (AF) (e.g., a firstantigen binding fragment (AF1), or a second antigen binding fragment(AF2)) can (each independently) be a chimeric or a humanized antigenbinding fragment. The antigen binding fragment (AF) (e.g., a firstantigen binding fragment (AF1), or a second antigen binding fragment(AF2)) can (each independently) be Fv, Fab, Fab′, Fab′-SH, linearantibody, or single-chain variable fragment (scFv). Two antigen bindingfragments (e.g., the first and second antigen binding fragments) can beconfigured as an (Fab′)2 or a single chain diabody. In some embodiments,the bispecific comprises a first binding domain with binding specificityto a target cell marker and a second binding domain with bindingspecificity to an effector cell antigen. In some embodiments, the firstand the second binding domains can be non-antibody scaffolds such asanticalins, adnectins, fynomers, affilins, affibodies, centyrins, orDARPins. In other embodiments, the binding domain for the tumor celltarget is a variable domain of a T cell receptor that has beenengineered to bind MHC that is loaded with a peptide fragment of aprotein that is overexpressed by tumor cells. In some embodiments, theXPAT compositions are designed with considerations of the location ofthe target tissue protease as well as the presence of the same proteasein healthy tissues not intended to be targeted, as well as the presenceof the target ligand in healthy tissue but a greater presence of theligand in unhealthy target tissue, in order to provide a widetherapeutic window. A “therapeutic window” refers to the largestdifference between the minimal effective dose and the maximal tolerateddose for a given therapeutic composition. To help achieve a widetherapeutic window, the binding domains of the first portion of thecompositions are shielded by the proximity of the bulking moiety (e.g.,XTEN) such that the binding affinity of the intact composition for oneor both of the ligands is reduced compared to the composition that hasbeen cleaved by a mammalian protease, thereby releasing the firstportion from the shielding effects of the bulking moiety.

With respect to single chain binding domains, as is well established, Fvis the minimum antibody fragment which contains a complete antigenrecognition and binding site; consisting of a dimer of one heavy (VH)and one light chain variable domain (VL) in non-covalent association.Within each VH and VL chain are three complementarity determiningregions (CDRs) that interact to define an antigen binding site on thesurface of the VH-VL dimer; the six CDRs of a binding domain conferantigen binding specificity to the antibody or single chain bindingdomain. In some cases, scFv are created in which each has 3, 4, or 5CHRs within each binding domain. Framework sequences flanking the CDRshave a tertiary structure that is essentially conserved in nativeimmunoglobulins across species, and the framework residues (FR) serve tohold the CDRs in their appropriate orientation. The constant domains arenot required for binding function, but may aid in stabilizing VH-VLinteraction. In some embodiments, the domain of the binding site of thepolypeptide can be a pair of VH-VL, VH-VH or VL-VL domains either of thesame or of different immunoglobulins, however it is generally preferredto make single chain binding domains using the respective VH and VLchains from the parental antibody. The order of VH and VL domains withinthe polypeptide chain is not limiting for the present invention; theorder of domains given may be reversed usually without any loss offunction, but it is understood that the VH and VL domains are arrangedso that the antigen binding site can properly fold. Thus, the singlechain binding domains of the bispecific scFv embodiments of the subjectcompositions can be in the order (VL-VH)1-(VL-VH)2, wherein “1” and “2”represent the first and second binding domains, respectively, or(VL-VH)1-(VH-VL)2, or (VH-VL)1-(VL-VH)2, or (VH-VL)1-(VH-VL)2, whereinthe paired binding domains are linked by a polypeptide linker asdescribed herein, below.

The arrangement of the binding domains in an exemplary bispecific singlechain antibody disclosed herein may therefore be one in which the firstbinding domain is located C-terminally to the second binding domain. Thearrangement of the V chains may be VH (target cell surface antigen)-VL(target cell surface antigen)-VL (effector cell antigen)-VH (effectorcell antigen), VH (target cell surface antigen)-VL (target cell surfaceantigen)-VH (effector cell antigen)-VL (effector cell antigen), VL(target cell surface antigen)-VH (target cell surface antigen)-VL(effector cell antigen)-VH (effector cell antigen) or VL (target cellsurface antigen)-VH (target cell surface antigen)-VH (effector cellantigen)-VL (effector cell antigen). For an arrangement, in which thesecond binding domain is located N-terminally to the first bindingdomain, the following orders are possible: VH (effector cell antigen)-VL(effector cell antigen)-VL (target cell surface antigen)-VH (target cellsurface antigen), VH (effector cell antigen)-VL (effector cellantigen)-VH (target cell surface antigen)-VL (target cell surfaceantigen), VL (effector cell antigen)-VH (effector cell antigen)-VL(target cell surface antigen)-VH (target cell surface antigen) or VL(effector cell antigen)-VH (effector cell antigen)-VH (target cellsurface antigen)-VL (target cell surface antigen). As used herein,“N-terminally to” or “C-terminally to” and grammatical variants thereofdenote relative location within the primary amino acid sequence ratherthan placement at the absolute N- or C-terminus of the bispecific singlechain antibody. Hence, as a non-limiting example, a first binding domainwhich is “located C-terminally to the second binding domain” denotesthat the first binding is located on the carboxyl side of the secondbinding domain within the bispecific single chain antibody, and does notexclude the possibility that an additional sequence, for example aHis-tag, or another compound such as a radioisotope, is located at theC-terminus of the bispecific single chain antibody.

In one embodiment, the chimeric polypeptide assembly compositionscomprise a first portion comprising a first binding domain and a secondbinding domain wherein each of the binding domains is an scFv andwherein each scFv comprises one VL and one VH. In some embodiments, thechimeric polypeptide assembly compositions comprise a first portioncomprising a first binding domain and a second binding domain whereinthe binding domains are in a diabody configuration and wherein eachdomain comprises one VL domain and one VH.

It is envisaged that the scFv embodiments of the XPAT compositions ofthe invention comprise a first binding domain and a second bindingdomain wherein the VL and VH domains are derived from monoclonalantibodies with binding specificity to the tumor-specific marker or anantigen of a target cell and effector cell antigens, respectively. Inother cases, the first and second binding domains each comprise six CDRsderived from monoclonal antibodies with binding specificity to a targetcell marker, such as a tumor-specific marker and effector cell antigens,respectively. In other embodiments, the first and second binding domainsof the first portion of the subject compositions can have 3, 4, or 5CHRs within each binding domain. In other embodiments, the embodimentsof the invention comprise a first binding domain and a second bindingdomain wherein each comprises a CDR-H1 region, a CDR-H2 region, a CDR-H3region, a CDR-L1 region, a CDR-L2 region, and a CDR-H3 region, whereineach of the regions is derived from a monoclonal antibody capable ofbinding the tumor-specific marker or an antigen of a target cell, andeffector cell antigens, respectively. In one embodiment, the inventionprovides a chimeric polypeptide assembly composition wherein the secondbinding domain comprises VH and VL regions derived from a monoclonalantibody capable of binding human CD3. In some embodiments, theinvention provides a chimeric polypeptide assembly composition, whereinthe scFv second binding domain comprises VH and VL regions wherein eachVH and VL regions exhibit at least about 90%, or 91%, or 92%, or 93%, or94%, or 95%, or 96%, or 97%, or 98%, or 99% identity to or is identicalto paired VL and VH sequences of an anti-CD3 antibody identified inTable 6a. In some embodiments, the second domain embodiments of theinvention comprise a CDR-H1 region, a CDR-H2 region, a CDR-H3 region, aCDR-L1 region, a CDR-L2 region, and a CDR-H3 region, wherein each of theregions is derived from a monoclonal antibody identified herein as theantibodies set forth in Table 6a. In the foregoing embodiments, the VHand/or VL domains can be configured as scFv, diabodies, a single domainantibody, or a single domain camelid antibody.

In other embodiments, the second domains of the subject compositions arederived from an anti-CD3 antibody identified herein as the antibodiesset forth in Table 6a. In one embodiment of the foregoing, the seconddomain of the subject composition comprises the paired VL and the VHregion sequences of the anti-CD3 antibody identified herein as the groupof antibodies set forth in Table 6a. In some embodiments, the inventionprovides a chimeric polypeptide assembly composition, wherein the secondbinding domain comprises VH and VL regions wherein each VH and VLregions exhibit at least about 90%, or 91%, or 92%, or 93%, or 94%, or95%, or 96%, or 97%, or 98%, or 99% identity to or is identical topaired VL and VH sequences of the huUCHT1 anti-CD3 antibody of Table 6a.In the foregoing embodiments, the VH and/or VL domains can be configuredas scFv, a portion of a diabody, a single domain antibody, or a singledomain camelid antibody.

In other embodiments, the scFv of the first domain of the compositionare derived from an anti-tumor cell antibody identified as theantibodies set forth in Table 6f. In some embodiments, the inventionprovides a chimeric polypeptide assembly composition, wherein the firstbinding domain comprises VH and VL regions wherein each VH and VLregions exhibit at least about 90%, or 91%, or 92%, or 93%, or 94%, or95%, or 96%, or 97%, or 98%, or 99% identity to or is identical topaired VL and VH sequences of an anti-tumor cell antibody identified inTable 6f. In one embodiment of the foregoing, the first domain of therecited compositions comprises the paired VL and the VH region sequencesof an anti-tumor cell antibody disclosed herein. In the foregoingembodiments, the VH and/or VL domains can be configured as scFv, aportion of a diabody, a single domain antibody, or a single domaincamelid antibody.

In some embodiments, the chimeric polypeptide assembly compositionscomprise a first portion comprising a first binding domain and a secondbinding domain wherein the binding domains are in a diabodyconfiguration and each of the binding domains comprises one VL domainand one VH domain. In one embodiment, the diabody embodiments of theinvention comprise a first binding domain and a second binding domainwherein the VL and VH domains are derived from monoclonal antibodieswith binding specificity to a tumor-specific marker or an antigen of atarget cell, and the effector cell antigen, respectively. In someembodiments, the diabody embodiments of the invention comprise a firstbinding domain and a second binding domain wherein each comprises aCDR-H1 region, a CDR-H2 region, a CDR-H3 region, a CDR-L1 region, aCDR-L2 region, and a CDR-H3 region, wherein each of the regions isderived from a monoclonal antibody capable of binding the tumor-specificmarker or target cell antigen, and the effector cell antigen,respectively. It is envisaged that the diabody embodiments of theinvention comprise a first binding domain and a second binding domainwherein the VL and VH domains are derived from monoclonal antibodieswith binding specificity to the tumor-specific marker or target cellantigen, and the effector cell antigen, respectively. In someembodiments, the diabody embodiments of the invention comprise a firstbinding domain and a second binding domain wherein each comprises aCDR-H1 region, a CDR-H2 region, a CDR-H3 region, a CDR-L1 region, aCDR-L2 region, and a CDR-H3 region, wherein each of the regions isderived from a monoclonal antibody capable of binding the tumor-specificmarker or target cell antigen, and the effector cell antigen,respectively. In one embodiment, the invention provides a chimericpolypeptide assembly composition wherein the diabody second bindingdomain comprises the paired VH and VL regions derived from a monoclonalantibody capable of binding human CD3. In some embodiments, theinvention provides a chimeric polypeptide assembly composition, whereinthe diabody second binding domain comprises VH and VL regions whereineach VH and VL regions exhibit at least about 90%, or 91%, or 92%, or93%, or 94%, or 95%, or 96%, or 97%, or 98%, or 99% identity to or isidentical to paired VL and VH sequences of an anti-CD3 antibodyidentified in Table 6a. In some embodiments, the invention provides achimeric polypeptide assembly composition, wherein the diabody secondbinding domain comprises VH and VL regions wherein each VH and VLregions exhibit at least about 90%, or 91%, or 92%, or 93%, or 94%, or95%, or 96%, or 97%, or 98%, or 99% identity to or is identical to theVL and a VH sequence of the huUCHT1 antibody of Table 6a. In otherembodiments, the diabody second domain of the composition is derivedfrom an anti-CD3 antibody described herein. In some embodiments, theinvention provides a chimeric polypeptide assembly composition, whereinthe diabody first binding domain comprises VH and VL regions whereineach VH and VL regions exhibit at least about 90%, or 91%, or 92%, or93%, or 94%, or 95%, or 96%, or 97%, or 98%, or 99% identity to or isidentical to VL and VH sequences of an anti-tumor cell antibodyidentified in Table 6f. In other embodiments, the diabody first domainof the composition is derived from an anti-tumor cell antibody describedherein.

Methods to measure binding affinity and/or other biologic activity ofthe subject compositions of the invention can be those disclosed hereinor methods generally known in the art. For example, the binding affinityof a binding pair (e.g., antibody and antigen), denoted as K_(d), can bedetermined using various suitable assays including, but not limited to,radioactive binding assays, non-radioactive binding assays such asfluorescence resonance energy transfer and surface plasmon resonance(SPR, Biacore), and enzyme-linked immunosorbent assays (ELISA), kineticexclusion assay (KinExA®) or as described in the Examples. An increaseor decrease in binding affinity, for example of a chimeric polypeptideassembly which has been cleaved to remove a bulking moiety compared tothe chimeric polypeptide assembly with the bulking moiety attached, canbe determined by measuring the binding affinity of the chimericpolypeptide assembly to its target binding partner with and without thebulking moiety.

Measurement of half-life of a subject chimeric assembly can be performedby various suitable methods. For example, the half-life of a substancecan be determined by administering the substance to a subject andperiodically sampling a biological sample (e.g., biological fluid suchas blood or plasma or ascites) to determine the concentration and/oramount of that substance in the sample over time. The concentration of asubstance in a biological sample can be determined using varioussuitable methods, including enzyme-linked immunosorbent assays (ELISA),immunoblots, and chromatography techniques including high-pressureliquid chromatography and fast protein liquid chromatography. In somecases, the substance may be labeled with a detectable tag, such as aradioactive tag or a fluorescence tag, which can be used to determinethe concentration of the substance in the sample (e.g., a blood sampleor a plasma sample. The various pharmacokinetic parameters are thendetermined from the results, which can be done using software packagessuch as SoftMax Pro software, or by manual calculations known in theart.

In addition, the physicochemical properties of the chimeric polypeptideassembly compositions may be measured to ascertain the degree ofsolubility, structure and retention of stability. Assays of the subjectcompositions are conducted that allow determination of bindingcharacteristics of the binding domains towards a ligand, includingbinding dissociation constant (K_(d), K_(on) and K_(off)), the half-lifeof dissociation of the ligand-receptor complex, as well as the activityof the binding domain to inhibit the biologic activity of thesequestered ligand compared to free ligand (IC₅₀ values). The term“IC₅₀” refers to the concentration needed to inhibit half of the maximumbiological response of the ligand agonist, and is generally determinedby competition binding assays. The term “EC₅₀” refers to theconcentration needed to achieve half of the maximum biological responseof the active substance, and is generally determined by ELISA orcell-based assays, including the methods of the Examples describedherein.

Anti-CD3 Binding Domains

In some embodiments, the invention provides chimeric polypeptideassembly compositions comprising a binding domain of the first portionwith binding affinity to T cells. In one embodiment, the binding domainof the second portion comprises VL and VH derived from a monoclonalantibody that binds CD3. In some embodiments, the binding domaincomprises VL and VH derived from a monoclonal antibody to CD3 epsilonand/or CD3 delta. Exemplary, non-limiting examples of VL and VHsequences of monoclonal antibodies to CD3 are presented in Table 6a. Inone embodiment, the invention provides a chimeric polypeptide assemblycomprising a binding domain with binding affinity to CD3 comprisinganti-CD3 VL and VH sequences set forth in Table 6a. In some embodiments,the invention provides a chimeric polypeptide assembly comprising abinding domain of the first portion with binding affinity to CD3epsiloncomprising anti-CD3epsilon VL and VH sequences set forth in Table 6a. Insome embodiments, the invention provides a chimeric polypeptide assemblycomposition, wherein the scFv second binding domain of the first portioncomprises VH and VL regions wherein each VH and VL regions exhibit atleast about 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%,or 98%, or 99% identity to or is identical to paired VL and VH sequencesof the huUCHT1 anti-CD3 antibody of Table 6a. In some embodiments, theinvention provides a chimeric polypeptide assembly compositioncomprising a binding domain with binding affinity to CD3 comprising theCDR-L1 region, the CDR-L2 region, the CDR-L3 region, the CDR-H1 region,the CDR-H2 region, and the CDR-H3 region, wherein each is derived fromthe respective anti-CD3 VL and VH sequences set forth in Table 6a. Insome embodiments, the invention provides a chimeric polypeptide assemblycomposition comprising a binding domain with binding affinity to CD3comprising the CDR-L1 region, the CDR-L2 region, the CDR-L3 region, theCDR-H1 region, the CDR-H2 region, and the CDR-H3 region, wherein the CDRsequences are

(SEQ ID NO: 50) RASQDIRNYLN, (SEQ ID NO: 78) YTSRLESQQGNTLPWT,(SEQ ID NO: 79) GYSFTGYTMN, (SEQ ID NO: 80) LINPYKGVST, and(SEQ ID NO: 81) SGYYGDSDWYFDV.

The CD3 complex is a group of cell surface molecules that associateswith the T-cell antigen receptor (TCR) and functions in the cell surfaceexpression of TCR and in the signaling transduction cascade thatoriginates when a peptide:MHC ligand binds to the TCR. Typically, whenan antigen binds to the T-cell receptor, the CD3 sends signals throughthe cell membrane to the cytoplasm inside the T cell. This causesactivation of the T cell that rapidly divide to produce new T cellssensitized to attack the particular antigen to which the TCR wereexposed. The CD3 complex is comprised of the CD3epsilon molecule, alongwith four other membrane-bound polypeptides (CD3-gamma, -delta, and/or-zeta). In humans, CD3-epsilon is encoded by the CD3E gene on Chromosome11. The intracellular domains of each of the CD3 chains containimmunoreceptor tyrosine-based activation motifs (ITAMs) that serve asthe nucleating point for the intracellular signal transduction machineryupon T cell receptor engagement.

A number of therapeutic strategies modulate T cell immunity by targetingTCR signaling, particularly the anti-human CD3 monoclonal antibodies(mAbs) that are widely used clinically in immunosuppressive regimes. TheCD3-specific mouse mAb OKT3 was the first mAb licensed for use in humans(Sgro, C. Side-effects of a monoclonal antibody, muromonabCD3/orthoclone OKT3: bibliographic review. Toxicology 105:23-29, 1995)and is widely used clinically as an immunosuppressive agent intransplantation (Chatenoud, Clin. Transplant 7:422-430, (1993);Chatenoud, Nat. Rev. Immunol. 3:123-132 (2003); Kumar, Transplant. Proc.30:1351-1352 (1998)), type 1 diabetes, and psoriasis. Importantly,anti-CD3 mAbs can induce partial T cell signaling and clonal anergy(Smith, JA, Nonmitogenic Anti-CD3 Monoclonal Antibodies Deliver aPartial T Cell Receptor Signal and Induce Clonal Anergy J. Exp. Med.185:1413-1422 (1997)). OKT3 has been described in the literature as a Tcell mitogen as well as a potent T cell killer (Wong, JT. The mechanismof anti-CD3 monoclonal antibodies. Mediation of cytolysis by inter-Tcell bridging. Transplantation 50:683-689 (1990)). In particular, thestudies of Wong demonstrated that by bridging CD3 T cells and targetcells, one could achieve killing of the target and that neitherFcR-mediated ADCC nor complement fixation was necessary for bivalentanti-CD3 MAB to lyse the target cells.

OKT3 exhibits both a mitogenic and T-cell killing activity in atime-dependent fashion; following early activation of T cells leading tocytokine release, upon further administration OKT3 later blocks allknown T-cell functions. It is due to this later blocking of T cellfunction that OKT3 has found such wide application as animmunosuppressant in therapy regimens for reduction or even abolition ofallograft tissue rejection. Other antibodies specific for the CD3molecule are disclosed in Tunnacliffe, Int. Immunol. 1 (1989), 546-50,WO2005/118635 and WO2007/033230 describe anti-human monoclonal CD3epsilon antibodies, U.S. Pat. No. 5,821,337 describes the VL and VHsequences of murine anti-CD3 monoclonal Ab UCHT1 (muxCD3, Shalaby etal., J. Exp. Med. 175, 217-225 (1992) and a humanized variant of thisantibody (hu UCHT1), and United States Patent Application 20120034228discloses binding domains capable of binding to an epitope of human andnon-chimpanzee primate CD3 epsilon chain.

TABLE 6a Anti-CD3 Monoclonal Antibodies and Sequences Clone AntibodySEQ ID SEQ ID Name Name Target VH Sequence NO. VL Sequence NO. huOKT3CD3 QVQLVQSGGGVVQP 301 DIQMTQSPSSLSASV 351 GRSLRLSCKAS GYT GDRVTITCSASSSVS FTRYTMH WVRQAP YMN WYQQTPGKAP GKGLEWIG YINPSR KRWIY DTSKLAS GVGYTNYNQKVKD RF PSRFSGSGSGTDYTF TISRDNSKNTAFLQ TISSLQPEDIATYYCMDSLRPEDTGVYFC QQWSSNPFT FGQGT AR YYDDHYCLDY W KLQITR GQGTPVTVSS huUCHT1CD3 EVQLVESGGGLVQP 302 DIQMTQSPSSLSASV 352 GGSLRLSCAAS GYS GDRVTITCRASQDIR FTGYTMN WVRQAP NYLN WYQQKPGKA GKGLEWVA LINPYK PKLLIY YTSRLES GVGVST YNQKFKDRFT PSRFSGSGSGTDYTL ISVDKSKNTAYLQM TISSLQPEDFATYYCNSLRAEDTAVYYCA QQGNTLPWT FGQG R SGYYGDSDWYFD TKVEIK V WGQGTLVTVSS hu12F6CD3 QVQLVQSGGGVVQP 303 DIQMTQSPSSLSASV 353 GRSLRLSCKAS GYT GDRVTMTCRASSSV FTSYTMH WVRQAP SYMH WYQQTPGKA GKGLEWIG YINPSS PKPWIY ATSNLAS GGYTKYNQKFKD RF VPSRFSGSGSGTDYT TISADKSKSTAFLQM LTISSLQPEDIATYYCDSLRPEDTGVYFCA QQWSSNPPT FGQGT R WQDYDVYFDY W KLQITR GQGTPVTVSS mOKT3CD3 QVQLQQSGAELARP 304 QIVLTQSPAIMSASP 354 GASVKMSCKAS GY GEKVTMTCSASSSV TFTRYTMH WVKQR SYMN WYQQKSGTS PGQGLEWIG YINPSR PKRWIY DTSKLAS GGYTNYNQKFKD KA VPAHFRGSGSGTSYS TLTTDKSSSTAYMQ LTISGMEAEDAATYLSSLTSEDSAVYYCA YC QQWSSNPFT FGS R YYDDHYCLDY WG GTKLEINR QGTTLTVSSMT103 blinatumomab CD3 DIKLQQSGAELARP 305 DIQLTQSPAIMSASP 355GASVKMSCKTS GYT GEKVTMTC RASSSV FTRYTMH WVKQRP SYMN WYQQKSGTS GQGLEWIGYINPSR PKRWIY DTSKVAS G GYTNYNQKFKD KA VPYRFSGSGSGTSYS TLTTDKSSSTAYMQLTISSMEAEDAATY LSSLTSEDSAVYYCA YC QQWSSNPLT FG R YYDDHYCLDY WG AGTKLELKQGTTLTVSS MT110 solitomab CD3 DVQLVQSGAEVKKP 306 DIVLTQSPATLSLSP 356GASVKVSCKAS GYT GERATLSC RASQSV FTRYTMH WVRQAP SYMN WYQQKPGKA GQGLEWIGYINPSR PKRWIY DTSKVAS G GYTNYADSVKG RF VPARFSGSGSGTDYS TITTDKSTSTAYMELLTINSLEAEDAATYY SSLRSEDTATYYCA C QQWSSNPLT FGG R YYDDHYCLDY WG GTKVEIKQGTTVTVSS CD3.7 CD3 EVQLVESGGGLVQP 307 QTVVTQEPSLTVSPG 357GGSLKLSCAASGFTF GTVTLTCGSSTGAV NKYAMNWVRQAPG TSGYYPNWVQQKPKGLEWVARIRSKYN GQAPRGLIGGTKFL NYATYYADSVKDRF APGTPARFSGSLLGGTISRDDSKNTAYLQ KAALTLSGVQPEDE MNNLKTEDTAVYY AEYYCALWYSNRW CVRHGNFGNSYISYVFGGGTKLTVL WAYWGQGTLVTVS S CD3.8 CD3 EVQLVESGGGLVQP 308 QAVVTQEPSLTVSP358 GGSLRLSCAASGFTF GGTVTLTCGSSTGA NTYAMNWVRQAPG VTTSNYANWVQQKKGLEWVGRIRSKYN PGQAPRGLIGGTNK NYATYYADSVKGRF RAPGVPARFSGSLLGTISRDDSKNTLYLQ GKAALTLSGAQPED MNSLRAEDTAVYYC EAEYYCALWYSNLVRHGNFGNSYVSWF WVFGGGTKLTVL AYWGQGTLVTVSS CD3.9 CD3 EVQLLESGGGLVQP 309ELVVTQEPSLTVSPG 359 GGSLKLSCAASGFTF GTVTLTCRSSTGAV NTYAMNWVRQAPGTTSNYANWVQQKP KGLEWVARIRSKYN GQAPRGLIGGTNKR NYATYYADSVKDRFAPGTPARFSGSLLGG TISRDDSKNTAYLQ KAALTLSGVQPEDE MNNLKTEDTAVYYAEYYCALWYSNLW CVRHGNFGNSYVS VFGGGTKLTVL WFAYWGQGTLVTV SS CD3.10 CD3EVKLLESGGGLVQP 310 QAVVTQESALTTSP 360 KGSLKLSCAASGFTF GETVTLTCRSSTGANTYAMNWVRQAPG VTTSNYANWVQEKP KGLEWVARIRSKYN DHLFTGLIGGTNKRNYATYYADSVKDRF APGVPARFSGSLIGD TISRDDSQSILYLQM KAALTITGAQTEDENNLKTEDTAMYYC AIYFCALWYSNLWV VRHGNFGNSYVSWF FGGGTKLTVL AYWGQGTLVTVSS*underlined sequences, if present, are CDRs within the VL and VH

CD3 Cell Antigen Binding Fragments

In some embodiments, the disclosure relates to antigen binding fragments(AF1) having specific binding affinity for an effector cell antigen thatcan be incorporated into any of the subject composition embodimentsdescribed herein. In some cases, the effector cell antigen is expressedon the surface of an effector cell that is a plasma cell, a T cell, a Bcell, a cytokine induced killer cell (CIK cell), a mast cell, adendritic cell, a regulatory T cell (RegT cell), a helper T cell, amyeloid cell, or a NK cell.

Various AF1 that bind effector cell antigens have particular utility forpairing with an antigen binding fragment with binding affinity to HER2antigens associated with a diseased cell or tissue in compositionformats in order to effect cell killing of the diseased cell or tissue.Binding specificity can be determined by complementarity determiningregions, or CDRs, such as light chain CDRs or heavy chain CDRs. In manycases, binding specificity is determined by light chain CDRs and heavychain CDRs. A given combination of heavy chain CDRs and light chain CDRsprovides a given binding pocket that confers greater affinity and/orspecificity towards an effector cell antigen as compared to otherreference antigens. The resulting bispecific compositions, having afirst antigen binding fragment (AF1) to HER2 linked by a short, flexiblepeptide linker to a second antigen binding fragment (AF2) with bindingspecificity to an effector cell antigen are bispecific, with eachantigen binding fragment having specific binding affinity to theirrespective ligands. It will be understood that in such compositions, anAF2 directed against a HER2 of a disease tissue is used in combinationwith a AF1 directed towards an effector cell marker in order to bring aneffector cell in close proximity to the cell of a disease tissue inorder to effect the cytolysis of the cell of the diseased tissue.Further, the AF1 and AF2 are incorporated into the specifically designedpolypeptides comprising cleavable release segments and XTEN in order toconfer prodrug characteristics on the compositions that becomesactivated by release of the fused AF1 and AF2 upon the cleavage of therelease segments when in proximity to the disease tissue havingproteases capable of cleaving the release segments in one or morelocations in the release segment sequence.

In one embodiment, the AF1 of the subject compositions has bindingaffinity for an effector cell antigen expressed on the surface of a Tcell. In some embodiments, the AF1 of the subject compositions hasbinding affinity for CD3. In some embodiments, the AF1 of the subjectcompositions has binding affinity for a member of the CD3 complex, whichincludes in individual form or independently combined form all known CD3subunits of the CD3 complex; for example, CD3 epsilon, CD3 delta, CD3gamma, and CD3 zeta. In some embodiments, the AF1 has binding affinityfor CD3 epsilon, CD3 delta, CD3 gamma, or CD3 zeta.

The origin of the antigen binding fragments contemplated by thedisclosure can be derived from a naturally occurring antibody orfragment thereof, a non-naturally occurring antibody or fragmentthereof, a humanized antibody or fragment thereof, a synthetic antibodyor fragment thereof, a hybrid antibody or fragment thereof, or anengineered antibody or fragment thereof. Methods for generating anantibody for a given target marker are well known in the art. Forexample, the monoclonal antibodies may be made using the hybridomamethod first described by Kohler et al., Nature, 256:495 (1975), or maybe made by recombinant DNA methods (U.S. Pat. No. 4,816,567). Thestructure of antibodies and fragments thereof, variable regions of heavyand light chains of an antibody (VH and VL), single chain variableregions (scFv), complementarity determining regions (CDR), and domainantibodies (dAbs) are well understood. Methods for generating apolypeptide having a desired antigen binding fragment with bindingaffinity to a given antigen are known in the art.

It will be understood that use of the term “antigen binding fragments”for the composition embodiments disclosed herein is intended to includeportions or fragments of antibodies that retain the ability to bind theantigens that are the ligands of the corresponding intact antibody. Insuch embodiments, the antigen binding fragment can be, but is notlimited to, CDRs and intervening framework regions, variable orhypervariable regions of light and/or heavy chains of an antibody (VL,VH), variable fragments (Fv), Fab′ fragments, F(ab′)2 fragments, Fabfragments, single chain antibodies (scAb), VHH camelid antibodies,single chain variable fragment (scFv), linear antibodies, a singledomain antibody, complementarity determining regions (CDR), domainantibodies (dAbs), single domain heavy chain immunoglobulins of the BHHor BNAR type, single domain light chain immunoglobulins, or otherpolypeptides known in the art containing a fragment of an antibodycapable of binding an antigen. The antigen binding fragments havingCDR-H and CDR-L can be configured in a (CDR-H)-(CDR-L) or a(CDR-H)-(CDR-L) orientation, N-terminus to C-terminus. The VL and VH oftwo antigen binding fragments can also be configured in a single chaindiabody configuration; i.e., the VL and VH of the AF1 and AF2 configuredwith linkers of an appropriate length to permit arrangement as adiabody.

Various CD3 binding AF1 of the disclosure have been specificallymodified to enhance their stability in the polypeptide embodimentsdescribed herein. Protein aggregation of antibodies continues to be asignificant problem in their developability and remains a major area offocus in antibody production. Antibody aggregation can be triggered bypartial unfolding of its domains, leading to monomer-monomer associationfollowed by nucleation and aggregate growth. Although the aggregationpropensities of antibodies and antibody-based proteins can be affectedby the external experimental conditions, they are strongly dependent onthe intrinsic antibody properties as determined by their sequences andstructures. Although it is well known that proteins are only marginallystable in their folded states, it is often less well appreciated thatmost proteins are inherently aggregation-prone in their unfolded orpartially unfolded states, and the resulting aggregates can be extremelystable and long-lived. Reduction in aggregation propensity has also beenshown to be accompanied by an increase in expression titer, showing thatreducing protein aggregation is beneficial throughout the developmentprocess and can lead to a more efficient path to clinical studies. Fortherapeutic proteins, aggregates are a significant risk factor fordeleterious immune responses in patients, and can form via a variety ofmechanisms. Controlling aggregation can improve protein stability,manufacturability, attrition rates, safety, formulation, titers,immunogenicity, and solubility. The intrinsic properties of proteinssuch as size, hydrophobicity, electrostatics and charge distributionplay important roles in protein solubility. Low solubility oftherapeutic proteins due to surface hydrophobicity has been shown torender formulation development more difficult and may lead to poorbio-distribution, undesirable pharmacokinetics behavior andimmunogenicity in vivo. Decreasing the overall surface hydrophobicity ofcandidate monoclonal antibodies can also provide benefits and costsavings relating to purification and dosing regimens. Individual aminoacids can be identified by structural analysis as being contributory toaggregation potential in an antibody, and can be located in CDR as wellas framework regions. In particular, residues can be predicted to be athigh risk of causing hydrophobicity issues in a given antibody. In oneembodiment, the present disclosure provides an AF1 having the capabilityto specifically bind CD3 in which the AF1 has at least one amino acidsubstitution of a hydrophobic amino acid in a framework region relativeto the parental antibody or antibody fragment wherein the hydrophobicamino acid is isoleucine, leucine or methionine. In some embodiments,the CD3 AF1 has at least two amino acid substitutions of hydrophobicamino acids in one or more framework regions wherein the hydrophobicamino acids are isoleucine, leucine or methionine.

The isoelectric point (pI) is the pH at which the antibody or antibodyfragment has no net electrical charge. If the pH is below the pI of anantibody or antibody fragment, then it will have a net positive charge.A greater positive charge tends to correlate with increased bloodclearance and tissue retention, with a generally shorter half-life. Ifthe pH is greater than the pI of an antibody or antibody fragment itwill have a negative charge. A negative charge generally results indecreased tissue uptake and a longer half-life. It is possible tomanipulate this charge through mutations to the framework residues.These considerations informed the design of the sequences of the AF1 ofthe embodiments described herein wherein individual amino acidsubstitutions were made relative to the parental antibody utilized asthe starting point. The isoelectric point of a polypeptide can bedetermined mathematically (e.g., computationally) or experimentally inan in vitro assay. The isoelectric point (pI) is the pH at which aprotein has a net charge of zero and can be calculated using the chargesfor the specific amino acids in the protein sequence. Estimated valuesfor the charges are called acid dissociation constants or pKa values andare used to calculate the pI. The pI can be determined in vitro bymethods such as capillary isoelectric focusing (see Datta-Mannan, A., etal. The interplay of non-specific binding, target-mediated clearance andFcRn interactions on the pharmacokinetics of humanized antibodies. mAbs7:1084 (2015); Li, B., et al. Framework selection can influencepharmacokinetics of a humanized therapeutic antibody through differencesin molecule charge. mAbs 6, 1255-1264 (2014)) or other methods known inthe art. In some embodiments, the isoelectric points of the AF1 and AF2are designed to be within a particular range of each other, therebypromoting stability.

In one embodiment, the present disclosure provides an antigen bindingfragment (e.g., AF1 or AF2) for use in any of the polypeptideembodiments described herein comprising CDR-L and CDR-H (see Table 6b),wherein the antigen binding fragment (e.g., AF1 or AF2) (a) specificallybinds to cluster of differentiation 3 T cell receptor (CD3); and (b)comprises CDR-H1, CDR-H2, and CDR-H3, having amino acid sequences of SEQID NOS: 8, 9, and 10, respectively. In some embodiments, the CDR-H1 andthe CDR-H2 of the antigen binding fragment (AF) can comprise amino acidsequences of SEQ ID NOS: 8 and 9, respectively. In some embodiments, thepresent disclosure provides an antigen binding fragment (e.g., AF1 orAF2) for use in any of the polypeptide embodiments described hereincomprising CDR-L and CDR-H, wherein the antigen binding fragment (e.g.,AF1 or AF2) (a) specifically binds to cluster of differentiation 3 Tcell receptor (CD3); (b) comprises CDR-H1, CDR-H2, and CDR-H3, havingamino acid sequences of SEQ ID NOS: 8, 9, and 10, respectively. Theantigen binding fragment (e.g., AF1 or AF2) can comprise CDR-L, whereinthe CDR-L comprises a CDR-L1 having an amino acid sequence of SEQ IDNOS: 1 or 2, a CDR-L2 having an amino acid sequence of SEQ ID NOS: 4 or5, and a CDR-L3 having an amino acid sequence of SEQ ID NO:6. In someembodiments, where the peptide comprises an antigen binding fragment(AF) (e.g., AF1 or AF2) comprising a CDR-L1, a CDR-L2, and a CDR-L3, theCDR-L1 of the AF can comprise an amino acid sequence of SEQ ID NO:1 or2; the CDR-L2 of the AF can comprise an amino acid sequence of SEQ IDNO: 4 or 5; and said CDR-L3 of the AF can comprise an amino acidsequence of SEQ ID NO:6. In some embodiments, where the peptidecomprises an antigen binding fragment (AF) (e.g., AF1 or AF2) comprisinga CDR-L1, a CDR-L2, and a CDR-L3, the CDR-L1 of the AF can comprise anamino acid sequence of SEQ ID NO:1; the CDR-L2 of the AF can comprise anamino acid sequence of SEQ ID NO: 4 or 5; and said CDR-L3 of the AF cancomprise an amino acid sequence of SEQ ID NO:6. In some embodiments,where the peptide comprises an antigen binding fragment (AF) (e.g., AF1or AF2) comprising a CDR-L1, a CDR-L2, and a CDR-L3, the CDR-L1 of theAF can comprise an amino acid sequence of SEQ ID NO: 2; the CDR-L2 ofthe AF can comprise an amino acid sequence of SEQ ID NO: 4 or 5; andsaid CDR-L3 of the AF can comprise an amino acid sequence of SEQ ID NO:6. In some embodiments, where the peptide comprises an antigen bindingfragment (AF) (e.g., AF1 or AF2) comprising a CDR-L1, a CDR-L2, and aCDR-L3, the CDR-L1 of the AF can comprise an amino acid sequence of SEQID NO: 1; the CDR-L2 of the AF can comprise an amino acid sequence ofSEQ ID NO: 4; and said CDR-L3 of the AF can comprise an amino acidsequence of SEQ ID NO: 6. In some embodiments, where the peptidecomprises an antigen binding fragment (AF) (e.g., AF1 or AF2) comprisinga CDR-L1, a CDR-L2, and a CDR-L3, the CDR-L1 of the AF can comprise anamino acid sequence of SEQ ID NO: 2; the CDR-L2 of the AF can comprisean amino acid sequence of SEQ ID NO: 5; and said CDR-L3 of the AF cancomprise an amino acid sequence of SEQ ID NO:6.

In some embodiments, the foregoing antigen binding fragment (AF) (e.g.,AF1 or AF2) embodiments of the immediately preceding paragraph furthercomprises light chain framework regions (FR-L) and heavy chain frameworkregions (FR-H) (see Table 6c) wherein the antigen binding fragment (AF)can comprise a FR-L1 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identicalto the amino acid sequence of SEQ ID NO:51, a FR-L2 exhibiting at least86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%sequence identity or is identical to the amino acid sequence of SEQ IDNO:52, a FR-L3 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical tothe amino acid sequence of any one of SEQ ID NOS: 53-56, a FR-L4exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% sequence identity or is identical to the amino acidsequence of SEQ ID NO: 59. The antigen binding fragment (AF) cancomprise a FR-L1 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical tothe amino acid sequence of SEQ ID NO:51, a FR-L2 exhibiting at least86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%sequence identity or is identical to the amino acid sequence of SEQ IDNO:52, a FR-L3 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical tothe amino acid sequence of SEQ ID NO: 53, a FR-L4 exhibiting at least86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%sequence identity or is identical to the amino acid sequence of SEQ IDNO: 59. The antigen binding fragment (AF) can comprise a FR-L1exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% sequence identity or is identical to the amino acidsequence of SEQ ID NO:51, a FR-L2 exhibiting at least 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identityor is identical to the amino acid sequence of SEQ ID NO:52, a FR-L3exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% sequence identity or is identical to the amino acidsequence of SEQ ID NO: 54, a FR-L4 exhibiting at least 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identityor is identical to the amino acid sequence of SEQ ID NO: 59. The antigenbinding fragment (AF) can comprise a FR-L1 exhibiting at least 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequenceidentity or is identical to the amino acid sequence of SEQ ID NO:51, aFR-L2 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% sequence identity or is identical to the aminoacid sequence of SEQ ID NO:52, a FR-L3 exhibiting at least 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequenceidentity or is identical to the amino acid sequence of SEQ ID NO: 55, aFR-L4 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% sequence identity or is identical to the aminoacid sequence of SEQ ID NO: 59. The antigen binding fragment (AF) cancomprise a FR-L1 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical tothe amino acid sequence of SEQ ID NO:51, a FR-L2 exhibiting at least86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%sequence identity or is identical to the amino acid sequence of SEQ IDNO:52, a FR-L3 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical tothe amino acid sequence of SEQ ID NO: 56, a FR-L4 exhibiting at least86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%sequence identity or is identical to the amino acid sequence of SEQ IDNO: 59. The antigen binding fragment (AF) can comprise a FR-H1exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% sequence identity or is identical to the amino acidsequence of any one of SEQ ID NOS: 60-63, a FR-H2 exhibiting at least86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%sequence identity or is identical to the amino acid sequence of SEQ IDNO: 64, a FR-H3 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical tothe amino acid sequence of SEQ ID NO: 65 or 66; and a FR-H4 exhibitingat least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% sequence identity or is identical to the amino acid sequence ofSEQ ID NO: 67. The antigen binding fragment (AF) can comprise a FR-H1exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% sequence identity or is identical to the amino acidsequence of SEQ ID NO: 60, a FR-H2 exhibiting at least 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identityor is identical to the amino acid sequence of SEQ ID NO: 64, a FR-H3exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% sequence identity or is identical to the amino acidsequence of SEQ ID NO: 65; and a FR-H4 exhibiting at least 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequenceidentity or is identical to the amino acid sequence of SEQ ID NO: 67.The antigen binding fragment (AF) can comprise a FR-H1 exhibiting atleast 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% sequence identity or is identical to the amino acid sequence of SEQID NO: 61, a FR-H2 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identicalto the amino acid sequence of SEQ ID NO: 64, a FR-H3 exhibiting at least86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%sequence identity or is identical to the amino acid sequence of SEQ IDNO: 65; and a FR-H4 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identicalto the amino acid sequence of SEQ ID NO: 67. The antigen bindingfragment (AF) (e.g., AF1 or AF2) for use in any of the polypeptideembodiments described herein can comprise light chain framework regions(FR-L) and heavy chain framework regions (FR-H) wherein the AF cancomprise a FR-L1 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical tothe amino acid sequence of SEQ ID NO: 51; a FR-L2 exhibiting at least86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%sequence identity or is identical to the amino acid sequence of SEQ IDNO: 52; a FR-L3 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical tothe amino acid sequence of any one of SEQ ID NOS: 53-56; a FR-L4exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% sequence identity or is identical to the amino acidsequence of SEQ ID NO: 59; a FR-H1 exhibiting at least 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identityor is identical to the amino acid sequence of SEQ ID NO: 60 or 61; aFR-H2 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% sequence identity or is identical to the aminoacid sequence of SEQ ID NO: 64; a FR-H3 exhibiting at least 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequenceidentity or is identical to the amino acid sequence of SEQ ID NO: 65;and a FR-H4 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to theamino acid sequence of SEQ ID NO: 67. The AF can comprise a FR-L1exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% sequence identity or is identical to the amino acidsequence of SEQ ID NO: 51; a FR-L2 exhibiting at least 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identityor is identical to the amino acid sequence of SEQ ID NO: 52; a FR-L3exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% sequence identity or is identical to the amino acidsequence of SEQ ID NO: 53; a FR-L4 exhibiting at least 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identityor is identical to the amino acid sequence of SEQ ID NO: 59; a FR-H1exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% sequence identity or is identical to the amino acidsequence of SEQ ID NO: 60; a FR-H2 exhibiting at least 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identityor is identical to the amino acid sequence of SEQ ID NO: 64; a FR-H3exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% sequence identity or is identical to the amino acidsequence of SEQ ID NO: 65; and a FR-H4 exhibiting at least 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequenceidentity or is identical to the amino acid sequence of SEQ ID NO: 67.The AF can comprise a FR-L1 exhibiting at least 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or isidentical to the amino acid sequence of SEQ ID NO: 51; a FR-L2exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% sequence identity or is identical to the amino acidsequence of SEQ ID NO: 52; a FR-L3 exhibiting at least 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identityor is identical to the amino acid sequence of SEQ ID NO: 54; a FR-L4exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% sequence identity or is identical to the amino acidsequence of SEQ ID NO: 59; a FR-H1 exhibiting at least 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identityor is identical to the amino acid sequence of SEQ ID NO: 61; a FR-H2exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% sequence identity or is identical to the amino acidsequence of SEQ ID NO: 64; a FR-H3 exhibiting at least 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identityor is identical to the amino acid sequence of SEQ ID NO: 65; and a FR-H4exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% sequence identity or is identical to the amino acidsequence of SEQ ID NO: 67. The AF can comprise a FR-L1 exhibiting atleast 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% sequence identity or is identical to the amino acid sequence of SEQID NO: 51; a FR-L2 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identicalto the amino acid sequence of SEQ ID NO: 52; a FR-L3 exhibiting at least86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%sequence identity or is identical to the amino acid sequence of SEQ IDNO: 55; a FR-L4 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical tothe amino acid sequence of SEQ ID NO: 59; a FR-H1 exhibiting at least86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%sequence identity or is identical to the amino acid sequence of SEQ IDNO: 61; a FR-H2 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical tothe amino acid sequence of SEQ ID NO: 64; a FR-H3 exhibiting at least86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%sequence identity or is identical to the amino acid sequence of SEQ IDNO: 65; and a FR-H4 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identicalto the amino acid sequence of SEQ ID NO: 67. The AF can comprise a FR-L1exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% sequence identity or is identical to the amino acidsequence of SEQ ID NO: 51; a FR-L2 exhibiting at least 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identityor is identical to the amino acid sequence of SEQ ID NO: 52; a FR-L3exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% sequence identity or is identical to the amino acidsequence of SEQ ID NO: 56; a FR-L4 exhibiting at least 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identityor is identical to the amino acid sequence of SEQ ID NO: 59; a FR-H1exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% sequence identity or is identical to the amino acidsequence of SEQ ID NO: 61; a FR-H2 exhibiting at least 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identityor is identical to the amino acid sequence of SEQ ID NO: 64; a FR-H3exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% sequence identity or is identical to the amino acidsequence of SEQ ID NO: 65; and a FR-H4 exhibiting at least 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequenceidentity or is identical to the amino acid sequence of SEQ ID NO: 67.

In some embodiments, the present disclosure provides an antigen bindingfragment (AF) (e.g., AF1 or AF2) for use in any of the polypeptideembodiments described herein wherein the AF comprises a variable heavy(VH) amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% sequence identity or is identical to an amino acidsequence of SEQ ID NO: 102 or SEQ ID NO: 105 of Table 6d. In someembodiments, the present disclosure provides an AF for use in any of thepolypeptide embodiments described herein wherein the AF comprises avariable light (VL) amino acid sequence having at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical toan amino acid sequence of any one of SEQ ID NOS: 101, 103, 104, 106, or107 of Table 6d. In some embodiment, the present disclosure provides anantigen binding fragment (e.g., AF1 or AF2) for use in any of thepolypeptide embodiments described herein wherein the AF can comprise anamino acid sequence having at least 95%, 96%, 97%, 98%, 99% sequenceidentity or is identical to an amino acid sequence of any one of SEQ IDNOS: 201-205 of Table 6e.

In some embodiments, the present disclosure provides antigen bindingfragment (e.g., AF1 or AF2) that bind to the CD3 protein complex thathave enhanced stability compared to CD3 binding antibodies or antigenbinding fragments known in the art. Additionally, the CD3 antigenbinding fragments of the disclosure are designed to confer a higherdegree of stability on the chimeric bispecific antigen binding fragmentcompositions into which they are integrated, leading to improvedexpression and recovery of the fusion protein, increased shelf-life andenhanced stability when administered to a subject. In one approach, theCD3 AF of the present disclosure are designed to have a higher degree ofthermal stability compared to certain CD3-binding antibodies and antigenbinding fragments known in the art. As a result, the CD3 AF utilized ascomponents of the chimeric bispecific antigen binding fragmentcompositions into which they are integrated exhibit favorablepharmaceutical properties, including high thermostability and lowaggregation propensity, resulting in improved expression and recoveryduring manufacturing and storage, as well promoting long serumhalf-life. Biophysical properties such as thermostability are oftenlimited by the antibody variable domains, which differ greatly in theirintrinsic properties. High thermal stability is often associated withhigh expression levels and other desired properties, including beingless susceptible to aggregation (Buchanan A, et al. Engineering atherapeutic IgG molecule to address cysteinylation, aggregation andenhance thermal stability and expression. MAbs 2013; 5:255). Thermalstability is determined by measuring the “melting temperature” (T_(m)),which is defined as the temperature at which half of the molecules aredenatured. The melting temperature of each heterodimer is indicative ofits thermal stability. In vitro assays to determine T_(m) are known inthe art, including methods described in the Examples, below. The meltingpoint of the heterodimer may be measured using techniques such asdifferential scanning calorimetry (Chen et al (2003) Pharm Res20:1952-60; Ghirlando et al (1999) Immunol Lett 68:47-52).Alternatively, the thermal stability of the heterodimer may be measuredusing circular dichroism (Murray et al. (2002) J. Chromatogr Sci40:343-9), or as described in the Examples, below.

In some embodiments of the polypeptides of this disclosure, the antigenbinding fragment (e.g., AF1 or AF2) can exhibit a higher thermalstability than an anti-CD3 binding fragment consisting of a sequence ofSEQ ID NO: 206 (see Table 6e), as evidenced in an in vitro assay by ahigher melting temperature (T_(m)) of the first antigen binding fragmentrelative to that of the anti-CD3 binding fragment; or upon incorporatingthe first antigen binding fragment into a test bispecific antigenbinding construct, a higher T_(m) of the test bispecific antigen bindingconstruct relative to that of a control bispecific antigen bindingconstruct, wherein the test bispecific antigen binding constructcomprises the first antigen binding fragment and a reference antigenbinding fragment that binds to an antigen other than CD3; and whereinthe control bispecific antigen binding construct consists of theanti-CD3 binding fragment consisting of the sequence of SEQ ID NO:206(see Table 6e) and the reference antigen binding fragment. The meltingtemperature (T_(m)) of the first antigen binding fragment can be atleast 2° C. greater, or at least 3° C. greater, or at least 4° C.greater, or at least 5° C. greater than the T_(m) of the anti-CD3binding fragment consisting of the sequence of SEQ ID NO: 206 (see Table6e).

Thermal denaturation curves of the CD3 binding fragments and theanti-CD3 bispecific antibodies comprising the anti-CD3 binding fragmentand a reference binding of the present disclosure show that theconstructs of the present disclosure are more resistant to thermaldenaturation than the antigen binding fragment consisting of a sequenceshown in SEQ ID NO: 781 (see Table 6f) or a control bispecific antibodywherein the control bispecific antigen binding fragment comprises SEQ IDNO: 781 (see Table 6f) and a reference antigen binding fragment thatbinds to a HER2 embodiment described herein. In one embodiment, thepolypeptides of any of the subject composition embodiments describedherein comprise an anti-CD3 AF of the embodiments described herein,wherein the T_(m) of the AF is at least 2° C. greater, or at least 3° C.greater, or at least 4° C. greater, or at least 5° C. greater, or atleast 6° C. greater, or at least 7° C. greater, or at least 8° C.greater, or at least 9° C. greater, or at least 10° C. greater than theT_(m) of an antigen binding fragment consisting of a sequence of SEQ IDNO: 781 (see Table 6f), as determined by an increase in meltingtemperature in an in vitro assay.

In some embodiments, the polypeptides of any of the subject compositionembodiments described herein comprise an antigen binding fragment (AF)that specifically bind human or cynomolgus monkey (cyno) CD3. Theantigen binding fragment (AF) can specifically bind human CD3. Theantigen binding fragment (AF) can bind a CD3 complex subunit identifiedherein as CD3 epsilon, CD3 delta, CD3 gamma, or CD3 zeta unit of CD3.The antigen binding fragment (AF) can bind a CD3 epsilon fragment ofCD3. The antigen binding fragment (AF) can specifically bind human orcyno CD3 with a dissociation constant (K_(d)) constant between about 10nM and about 400 nM, or between about 50 nM and about 350 nM, or betweenabout 100 nM and 300 nM, as determined in an in vitro antigen-bindingassay comprising a human or cyno CD3 antigen. In some embodiments, thepolypeptides of any of the subject composition embodiments describedherein comprise an antigen binding fragment (AF) that specifically bindshuman or cyno CD3 with a dissociation constant (K_(d)) weaker than about10 nM, or about 50 nM, or about 100 nM, or about 150 nM, or about 200nM, or about 250 nM, or about 300 nM, or about 350 nM, or weaker thanabout 400 nM as determined in an in vitro antigen-binding assay. Forclarity, an antigen binding fragment (AF) with a K_(d) of 400 binds itsligand more weakly than one with a K_(d) of 10 nM. In some embodiments,the polypeptides of any of the subject composition embodiments describedherein comprise an antigen binding fragment (AF) that specifically bindshuman or cyno CD3 with at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold,7-fold, 8-fold, 9-fold, or at least 10-fold weaker binding affinity thanan antigen binding fragment consisting of an amino acid sequence of SEQID NO: 781 (see Table 6f), as determined by the respective dissociationconstants (K_(d)) in an in vitro antigen-binding assays. In someembodiments, the present disclosure provides bispecific polypeptidescomprising an antigen binding fragment (AF) that exhibits a bindingaffinity to CD3 (anti-CD3 AF) that is at least 2-fold, 3-fold, 4-fold,5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 50-fold,100-fold, or at least 1000-fold at weaker relative to that of ananti-HER2 AF embodiments described herein that are incorporated into thesubject polypeptides, as determined by the respective dissociationconstants (K_(d)) in an in vitro antigen-binding assay. The bindingaffinity of the subject compositions for the target ligands can beassayed using binding or competitive binding assays, such as Biacoreassays with chip-bound receptors or binding proteins or ELISA assays, asdescribed in U.S. Pat. No. 5,534,617, assays described in the Examplesherein, radio-receptor assays, or other assays known in the art. Thebinding affinity constant can then be determined using standard methods,such as Scatchard analysis, as described by van Zoelen, et al., TrendsPharmacol Sciences (1998) 19)12):487, or other methods known in the art.

In a related aspect, the present disclosure provides an antigen bindingfragment (AF) that bind to CD3 (anti-CD3 AF) and are incorporated intochimeric, bispecific polypeptide compositions that are designed to havean isoelectric point (pI) that confer enhanced stability on thecompositions of the disclosure compared to corresponding compositionscomprising CD3 binding antibodies or antigen binding fragments known inthe art. In one embodiment, the polypeptides of any of the subjectcomposition embodiments described herein comprise AF that bind to CD3(anti-CD3 AF) wherein the anti-CD3 AF exhibits a pI that is between 6.0and 6.6, inclusive. In some embodiments, the polypeptides of any of thesubject composition embodiments described herein comprise AF that bindto CD3 (anti-CD3 AF) wherein the anti-CD3 AF exhibits a pI that is atleast 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 pH unit lowerthan the pI of a reference antigen binding fragment (e.g., consisting ofa sequence shown in SEQ ID NO: 206 (see Table 6e)). In some embodiments,the polypeptides of any of the subject composition embodiments describedherein comprise an AF that binds to CD3 (anti-CD3 AF) fused to anotherAF that binds to a HER2 antigen (anti-HER2 AF) wherein the anti-CD3 AFexhibits a pI that is within at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7,0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, or 1.5 pH units of the pI of the AFthat binds HER2 antigen or an epitope thereof. In some embodiments, thepolypeptides of any of the subject composition embodiments describedherein comprise an AF that binds to CD3 (anti-CD3 AF) fused to an AFthat binds to a HER2 antigen (anti-HER2 AF) wherein the AF exhibits a pIthat is within at least about 0.1 to about 1.5, or at least about 0.3 toabout 1.2, or at least about 0.5 to about 1.0, or at least about 0.7 toabout 0.9 pH units of the pI of the anti-CD3 AF. It is specificallyintended that by such design wherein the pI of the two antigen bindingfragments are within such ranges, the resulting fused antigen bindingfragments will confer a higher degree of stability on the chimericbispecific antigen binding fragment compositions into which they areintegrated, leading to improved expression and enhanced recovery of thefusion protein in soluble, non-aggregated form, increased shelf-life ofthe formulated chimeric bispecific polypeptide compositions, andenhanced stability when the composition is administered to a subject.State differently, having the two AFs (the anti-CD3 AF and the anti-HER2AF) within a relatively narrow pI range of may allow for the selectionof a buffer or other solution in which both the AFs (anti-CD3 AF andanti-HER2 AF) are stable, thereby promoting overall stability of thecomposition. The antigen binding fragment (AF) can exhibit anisoelectric point (pI) that is less than or equal to 6.6. The antigenbinding fragment (AF) can exhibit an isoelectric point (pI) that isbetween 6.0 and 6.6, inclusive. The antigen binding fragment (AF) canexhibit an isoelectric point (pI) that is at least 0.1, 0.2, 0.3, 0.4,0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 pH units lower than the pI of areference antigen binding fragment consisting of a sequence shown in SEQID NO: 206 (see Table 6e). The antigen binding fragment (AF) canspecifically bind human or cyno CD3 with a dissociation constant (K_(d))constant between about between about 10 nM and about 400 nM (such asdetermined in an in vitro antigen-binding assay comprising a human orcyno CD3 antigen). The antigen binding fragment (AF) can specificallybind human or cyno CD3 with a dissociation constant (K_(d)) of less thanabout 10 nM, or less than about 50 nM, or less than about 100 nM, orless than about 150 nM, or less than about 200 nM, or less than about250 nM, or less than about 300 nM, or less than about 350 nM, or lessthan about 400 nM (such as determined in an in vitro antigen-bindingassay). The antigen binding fragment (AF) can exhibit a binding affinityto CD3 that is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold,8-fold, 9-fold, or at least 10-fold weaker relative to that of anantigen binding fragment consisting of an amino acid sequence of SEQ IDNO: 206 (see Table 6e) (such as determined by the respectivedissociation constants (K_(d)) in an in vitro antigen-binding assay).

In certain embodiments, the VL and VH of the antigen binding fragmentsare fused by relatively long linkers, consisting 25, 26, 27, 28, 29, 30,31, 32, 33, 34, or 35 hydrophilic amino acids that, when joinedtogether, have a flexible characteristic. In one embodiment, the VL andVH of any of the scFv embodiments described herein are linked byrelatively long linkers of hydrophilic amino acids having the sequencesGSGEGSEGEGGGEGSEGEGSGEGGEGEGSG (SEQ ID NO: 82),TGSGEGSEGEGGGEGSEGEGSGEGGEGEGSGT (SEQ ID NO: 83),GATPPETGAETESPGETTGGSAESEPPGEG (SEQ ID NO: 84), orGSAAPTAGTTPSASPAPPTGGSSAAGSPST (SEQ ID NO: 85). In some embodiments, theAF1 and AF2 are linked together by a short linker of hydrophilic aminoacids having 3, 4, 5, 6, or 7 amino acids. In one embodiment, the shortlinker sequences are identified herein as the sequences SGGGGS (SEQ IDNO: 86), GGGGS (SEQ ID NO: 87), GGSGGS (SEQ ID NO: 88), GGS, or GSP. Insome embodiments, the disclosure provides compositions comprising asingle chain diabody in which after folding, the first domain (VL or VH)is paired with the last domain (VH or VL) to form one scFv and the twodomains in the middle are paired to form the other scFv in which thefirst and second domains, as well as the third and last domains, arefused together by one of the foregoing short linkers and the second andthe third variable domains are fused by one of the foregoing relativelylong linkers. As will be appreciated by one of skill in the art, theselection of the short linker and relatively long linker is to preventthe incorrect pairing of adjacent variable domains, thereby facilitatingthe formation of the single chain diabody configuration comprising theVL and VH of the first antigen binding fragment and the second antigenbinding fragment.

TABLE 6b Exemplary CD3 CDR Sequences CDR Amino SEQ Construct REGIONAcid Sequence ID NO: 3.23, 3.30, 3.31, 3.32 CDR-L1 RSSNGAVTSSNYAN  13.24 CDR-L1 RSSNGEVTTSNYAN  2 3.33, 3.9 CDR-L1 RSSTGAVTTSNYAN  33.23, 3.30, 3.31, 3.32, 3.9, 3.33 CDR-L2 GTNKRAP  4 3.24 CDR-L2 GTIKRAP 5 3.23, 3.24, 3.30, 3.31, 3.32 CDR-L3 ALWYPNLWVF  6 3.33, 3.9 CDR-L3ALWYSNLWVF  7 3.23, 3.24, 3.30, 3.31, 3.32, 3.9, CDR-H1 GFTFNTYAMN  83.33 3.23, 3.24, 3.30, 3.31, 3.32, 3.9, CDR-H2 RIRSKYNNYATYYADSVKD  93.33 3.23. 3.24, 3.30, 3.31, 3.32 CDR-H3 HENFGNSYVSWFAH 10 3.9, 3.33CDR-H3 HGNFGNSYVSWFAY 11

TABLE 6c Exemplary CD3 FR Sequences FR SEQ ID Construct REGIONAmino Acid Sequence NO: 3.23, 3.24, 3.30, 3.31, FR-L1ELVVTQEPSLTVSPGGTVTLTC 51 3.32, 3.9, 3.33 3.23, 3.24, 3.30, 3.31, FR-L2WVQQKPGQAPRGLIG 52 3.32, 3.9, 3.33 3.23, 3.24 FR-L3GTPARFSGSLLGGKAALTLSGVQPEDEAVYYC 53 3.30 FR-L3GTPARFSGSSLGGKAALTLSGVQPEDEAVYYC 54 3.31 FR-L3GTPARFSGSLLGGSAALTLSGVQPEDEAVYYC 55 3.32 FR-L3GTPARFSGSSLGGSAALTLSGVQPEDEAVYYC 56 3.9 FR-L3GTPARFSGSLLGGKAALTLSGVQPEDEAEYYC 57 3.33 FR-L3GTPARFSGSSLGGSAALTLSGVQPEDEAEYYC 58 3.23, 3.24, 3.30, 3.31, FR-L4GGGTKLTVL 59 3.32, 3.9, 3.33 3.23, 3.24 FR-H1 EVQLLESGGGIVQPGGSLKLSCAAS60 3.30, 3.31, 3.32 FR-H1 EVQLQESGGGIVQPGGSLKLSCAAS 61 3.33 FR-H1EVQLQESGGGLVQPGGSLKLSCAAS 62 3.9 FR-H1 EVQLLESGGGLVQPGGSLKLSCAAS 633.23, 3.24, 3.30, 3.31, FR-H2 WVRQAPGKGLEWVA 64 3.32, 3.9, 3.333.23, 3.24, 3.30, 3.31, FR-H3 RFTISRDDSKNTVYLQMNNLKTEDTAVYYCVR 65 3.323.9, 3.33 FR-H3 RFTISRDDSKNTAYLQMNNLKTEDTAVYYCVR 663.23, 3.24, 3.30, 3.31, FR-H4 WGQGTLVTVSS 67 3.32, 3.9, 3.33

TABLE 6d Exemplary CD3 VL & VH Sequences SEQ ID Construct REGIONAmino Acid Sequence NO: 3.23 VLELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAP 101RGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAVYYCAL WYPNLWVFGGGTKLTVL3.23, 3.24 VH EVQLLESGGGIVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGL 102EWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTVYLQMNNLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSS 3.24 VLELVVTQEPSLTVSPGGTVTLTCRSSNGEVTTSNYANWVQQKPGQAP 103RGLIGGTIKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAVYYCAL WYPNLWVFGGGTKLTVL 3.30 VLELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAP 104RGLIGGTNKRAPGTPARFSGSSLGGKAALTLSGVQPEDEAVYYCAL WYPNLWVFGGGTKLTVL3.30, 3.31, VH EVQLQESGGGIVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGL 105 3.32EWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTVYLQMNNLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSS 3.31 VLELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAP 106RGLIGGTNKRAPGTPARFSGSLLGGSAALTLSGVQPEDEAVYYCAL WYPNLWVFGGGTKLTVL 3.32 VLELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAP 107RGLIGGTNKRAPGTPARFSGSSLGGSAALTLSGVQPEDEAVYYCAL WYPNLWVFGGGTKLTVL 3.9 VLELVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAP 108RGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCAL WYSNLWVFGGGTKLTVL 3.9 VHEVQLLESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGL 109EWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS 3.33 VLELVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAP 110RGLIGGTNKRAPGTPARFSGSSLGGSAALTLSGVQPEDEAEYYCAL WYSNLWVFGGGTKLTVL 3.33 VHEVQLQESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGL 111EWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS

TABLE 6e Exemplary CD3 scFv Sequences SEQ ID ConstructAmino Acid Sequence NO: 3.23ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNK 201RAPGTPARFSGSLLGGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVLGATPPETGAETESPGETTGGSAESEPPGEGEVQLLESGGGIVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTVYLQMNNLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSS 3.24ELVVTQEPSLTVSPGGTVTLTCRSSNGEVTTSNYANWVQQKPGQAPRGLIGGTIKR 202APGTPARFSGSLLGGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVLGATPPETGAETESPGETTGGSAESEPPGEGEVQLLESGGGIVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTVYLQMNNLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSS 3.30ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNK 203RAPGTPARFSGSSLGGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVLGATPPETGAETESPGETTGGSAESEPPGEGEVQLQESGGGIVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTVYLQMNNLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSS 3.31ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNK 204RAPGTPARFSGSLLGGSAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVLGATPPETGAETESPGETTGGSAESEPPGEGEVQLQESGGGIVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTVYLQMNNLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSS 3.32ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNK 205RAPGTPARFSGSSLGGSAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVLGATPPETGAETESPGETTGGSAESEPPGEGEVQLQESGGGIVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTVYLQMNNLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSS 3.9ELVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNK 206RAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVLGATPPETGAETESPGETTGGSAESEPPGEGEVQLLESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS 3.33ELVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNK 207RAPGTPARFSGSSLGGSAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVLGATPPETGAETESPGETTGGSAESEPPGEGEVQLQESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS 4.11QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYRNNQRPS 208GVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLSGLWVFGGGTKLTVLGATPPETGAETESPGETTGGSAESEPPGEGQVQLQQWGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSRINSDGSSTNYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCARELRWGNWGQGTLVTVSS 4.12QAGLTQPPSASGTPGQRVTLSCSGSYSNIGTYYVYWYQQLPGTAPKLLIYSNDQRL 209SGVPDRFSGSKSGTSASLAISGLQSEDEAAYYCAAWDDSLNGWAFGGGTKLTVLGATPPETGAETESPGETTGGSAESEPPGEGQVQLQQWGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSRINSDGSSTNYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCARELRWGNWGQGTLVTVSS 4.13QPGLTQPPSASGTPGQRVTLSCSGRSSNIGSYYVYWYQHLPGMAPKLLIYRNSRRP 210SGVPDRFSGSKSGTSASLVISGLQSDDEADYYCAAWDDSLKSWVFGGGTKLTVLGATPPETGAETESPGETTGGSAESEPPGEGQVQLQQWGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSRINSDGSSTNYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCARELRWGNWGQGTLVTVSS 4.14QSVLTQPPSASGTPGQRVTISCSGSSSNIGTNYVYWYQQFPGTAPKLLIYSNNQRPS 211GVPDRFSGSKSGTSGSLAISGLQSEDEADYSCAAWDDSLNGWVFGGGTKLTVLGATPPETGAETESPGETTGGSAESEPPGEGQVQLVQWGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSRINSDGSSTNYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCARELRWGNWGQGTLVTVSS 4.15QPGLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYRNNQRPS 212GVPDRLSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLSGWVFGGGTKLTVLGATPPETGAETESPGETTGGSAESEPPGEGQVQLVQWGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSRINSDGSSTNYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCARELRWGNWGQGTLVTVSS 4.16QAVLTQPPSASGTPGQRVTISCSGSSSNIGSYYVYWYQQVPGAAPKLLMRLNNQR 213PSGVPDRFSGAKSGTSASLVISGLRSEDEADYYCAAWDDSLSGQWVFGGGTKLTVLGATPPETGAETESPGETTGGSAESEPPGEGQVQLQQWGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSRINSDGSSTNYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCARELRWGNWGQGTLVTVSS 4.17QAGLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYRNNQRPS 214GVPDRFSGSKSGTSASLAISGLRSEDEADYYCATWDASLSGWVFGGGTKLTVLGATPPETGAETESPGETTGGSAESEPPGEGEVQLVQWGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSRINSDGSSTNYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCARELRWGNWGQGTLVTVSS

Anti-HER-2 Binding Domains

In some embodiments, the invention provides chimeric polypeptideassembly compositions comprising a first portion binding domain withbinding affinity to the tumor-specific marker HER-2 and a second bindingdomain binds to an effector cell antigen, such as CD3 antigen. In oneembodiment, the binding domain comprises VL and VH derived form amonoclonal antibody to HER-2. Monoclonal antibodies to HER-2 are knownin the art. Exemplary, non-limiting examples of VL and VH sequences arepresented in Table 6f. In one embodiment, the invention provides achimeric polypeptide assembly composition comprising a first portionbinding domain with binding affinity to the tumor-specific marker HER-2comprising anti-HER-2 VL and VH sequences set forth in Table 6f. In someembodiments, the invention provides a chimeric polypeptide assemblycomposition comprising a first portion binding domain with bindingaffinity to the tumor-specific marker comprising the CDR-L1 region, theCDR-L2 region, the CDR-L3 region, the CDR-H1 region, the CDR-H2 region,and the CDR-H3 region, wherein each is derived from the respective VLand VH sequences set forth in Table 6f. Preferably, in the embodiments,the binding has a K_(d) value of greater than 10⁻¹⁰ to 10⁻⁷ M, asdetermined in an vitro binding assay. In some embodiments, where thepolypeptide comprises an antigen binding fragment that specificallybinds to HER2 (anti-HER2 AF), the anti-HER2 AF (e.g., AF1 or AF2) cancomprise (1) a heavy chain variable region (VH_(II)) comprising an aminoacid sequence set forth as SEQ ID NOS: 778-783, and (2) a light chainvariable region (VL_(II)) comprising an amino acid sequence set forth asSEQ ID NOS: 878-883 of Table 6f. It is specifically contemplated thatthe chimeric polypeptide assembly composition can comprise any one ofthe foregoing binding domains or sequence variants thereof so long asthe variants exhibit binding specificity for the described antigen. Inone embodiment, a sequence variant would be created by substitution ofan amino acid in the VL or VH sequence with a different amino acid. Indeletion variants, one or more amino acid residues in a VL or VHsequence as described herein are removed. Deletion variants, therefore,include all fragments of a binding domain polypeptide sequence. Insubstitution variants, one or more amino acid residues of a VL or VH (orCDR) polypeptide are removed and replaced with alternative residues. Inone aspect, the substitutions are conservative in nature andconservative substitutions of this type are well known in the art. Inaddition, it is specifically contemplated that the compositionscomprising the first and the second binding domains disclosed herein canbe utilized in any of the methods disclosed herein.

TABLE 6f Anti-HER2 Monoclonal Antibodies and Sequences Trade AntibodySEQ ID SEQ ID Name Name Target VH Sequence NO: VL Sequence NO: C1 HER2QVQLVESGGGLVQP 778 QSPSFLSAFVGDRITI 878 GGSLRLSCAASGFTF TC RASPGIRNYLA WS SYAMG WVRQAPG YQQKPGKAPKLLIY KGLEWVS SISGSSR AASTL Q S GVPSRFSYIYYADSVKG RFTIS GSGSGTDFTLTISSL RDNSKNTLYLQMNS QPEDFATYYC Q QYNLRAEDTAVYYCAK SYPLS FGGGTKVEIK MDASGSYFNF WGQ GTLVTVSS Erbicin HER2QVQLLQSAAEVKKP 779 QAVVTQEPSFSVSPG 879 GESLKISCKGSGYSF GTVTLTC GLSSGSV TSYWIG WVRQMPG STSYYPS WYQQTPG KGLEWMG IIYPGDS QAPRTLIY STNTRSSDTRYSPSFQG QVTIS GVPDRFSGSILGNKA ADKSISTAYLQWSSL ALTITGAQADDESDKASDTAVYYCAR W YYC VLYMGSGQYV RDSPL WGQGTLVTV FGGGTKLTVL SS Herceptintrastuzumab HER2 EVQLVESGGGLVQP 780 DIQMTQSPSSLSASV 880 GGSLRLSCAAS GFNIGDRVTITCRAS QDV KDTY IHWVRQAPG NTA VAWYQQKPGK KGLEWVAR IYPTNG APKLLIYSAS FLYSG YTRYADSVKG RFTIS VPSRFSGSRSGTDFT ADTSKNTAYLQMNSLTISSLQPEDFATYY LRAEDTAVYYCS R C Q QHYTTPPT FGQG WGGDGFYAMDY W TKVEIKGQGTLVTVSS MAGH22 margetuximab HER2 QVQLQQSGPELVKP 781 DIVMTQSHKFMSTS881 GASLKLSCTAS GFNI VGDRVSITCKAS QD KDTY IHWVKQRPEQ VNTA VAWYQQKPGGLEWIGR IYPTNGY HSPKLLIY SAS FRYT TRYDPKFQD KATIT GVPDRFTGSRSGTDFADTSSNTAYLQVSR TFTISSVQAEDLAVY LTSEDTAVYYC SRW YC QQHYTTPPT FGGGGDGFYAMDY WG GTKVEIK QGASVTVSS MM-302 F5 HER2 QVQLVESGGGLVQP 782QSVLTQPPSVSGAPG 882 GGSLRLSCAASGFTF QRVTISC TGSSSNIG R SYAMS WVRQAPGAGYGVH WYQQLPG KGLEWVS AISGRGD TAPKLLIY GNTNRPS NTYYADSVKG RFTIGVPDRFSGFKSGTSA SRDNSKNTLYLQMN SLAITGLQAEDEAD SLRAEDTAVYYC AK YYCQFYDSSLSGW MTSNAFAFDY WGQ V FGGGTKLTVL GTLVTVSS Perjeta pertuzumab HER2EVQLVESGGGLVQP 783 DIQMTQSPSSLSASV 883 GGSLRLSCAAS GFT GDRVTITCKASQ DVFTDYT MDWVRQAP SIG VAWYQQKPGK GKGLEWVAD VNPN APKLLIY SAS YRYTG SGGSIYNQRFKGRFT VPSRFSGSGSGTDFT LSVDRSKNTLYLQM LTISSLQPEDFATYY NSLRAEDTAVYYCA C QQYYIYPYT FGQG RNLGPSFYFDY WG TKVEIK QGTLVTVSS *underlined & boldedsequences, if present, are CDRs within the VL and VH

TABLE A Intramolecular Long Linkers Linker # Name SEQ IDAmino Acid Sequence L1 (G4S)3 112 GGGGSGGGGSGGGGS L2 MT110_18 113GEGTSTGSGGSGGSGGAD L3 MT103_18 114 VEGGSGGSGGSGGSGGVD L4 UCHT1_29 115RTSGPGDGGKGGPGKGPGGEGTKGTGPGG L5 Y30 116 GSGEGSEGEGGGEGSEGEGSGEGGEGEGSGL6 Y32 117 TGSGEGSEGEGGGEGSEGEGSGEGGEGEGSGT L7 G1_30_3 118GATPPETGAETESPGETTGGSAESEPPGEG L8 G9_30_1 119GSAAPTAGTTPSASPAPPTGGSSAAGSPST L9 Y30_modified 120GEGGESGGSEGEGSGEGEGGSGGEGESEGG L10 G1_30_1 121STETSPSTPTESPEAGSGSGSPESPSGTEA L11 G1_30_2 122PTGTTGEPSGEGSEPEGSAPTSSTSEATPS L12 G1_30_4 123SESESEGEAPTGPGASTTPEPSESPTPETS L13 UCHT1_modified 124PEGGESGEGTGPGTGGEPEGEGGPGGEGGT

TABLE B Intermolecular Short Linkers Name Amino Acid Sequence S-1SGGGGS (SEQ ID NO: 86) S-2 GGGGS (SE ID NO: 87) S-3 GGS S-4 GSP

In some embodiments of the polypeptides of this disclosure, a pair ofthe light chain variable region (VL) and the heavy chain variable region(VH) of an antigen binding fragment can be linked by a linker, or a longlinker (e.g., of hydrophilic amino acids). Such linker, linking thelight chain variable region (VL) and the heavy chain variable region(VH) of an antigen binding fragment (e.g., a first antigen bindingfragment (AF1), a second antigen binding fragment (AF2)), can (eachindependently) comprise an amino acid sequence having at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to asequence set forth in Table A. Such linker, linking the light chainvariable region (VL) and the heavy chain variable region (VH) of anantigen binding fragment (e.g., a first antigen binding fragment (AF1),a second antigen binding fragment (AF2)), can (each independently)comprise an amino acid sequence identical to a sequence set forth inTable A. In some embodiments of the polypeptides of this disclosure, twoantigen binding fragments (e.g., a first and a second antigen bindingfragments) can be fused together by a peptide linker, or a short linker.Such peptide linker, linking two antigen binding fragments (e.g., afirst and a second antigen binding fragments), can comprise an aminoacid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or 100% sequence identity to a sequence set forth in Table B.Such peptide linker, linking two antigen binding fragments (e.g., afirst and a second antigen binding fragments), can comprise an aminoacid sequence identical to a sequence set forth in Table B. In somecases, the first antigen binding fragment is a single-chain variablefragment (scFv). In some cases, the second antigen binding fragment is asingle-chain variable fragment (scFv). The two single-chain variablefragments of the first and second antigen binding fragments can belinked together by the peptide linker. In some embodiments of thepolypeptides of this disclosure, the linker used to link the VL and VHof the first antigen binding fragment or/and the linker used to link theVL and VH of the second antigen binding fragment can be L7 of Table A.In such embodiments, the peptide linker used to link the two antigenbinding fragments can be S-1 or S-2 of Table B. In some embodiments, thedisclosure provides polypeptides comprising a single chain diabody inwhich after folding, the first domain (VL or VH) is paired with the lastdomain (VH or VL) to form one scFv and the two domains in the middle arepaired to form the other scFv in which the first and second domains, aswell as the third and last domains, are fused together by a short linkerof hydrophilic amino acids identified herein by the sequences set forthin Table B and the second and the third variable domains are fused by along linker identified in Table A. As will be appreciated by one ofskill in the art, the selection of the short linker and long linker isto prevent the incorrect pairing of adjacent variable domains, therebyfacilitating the formation of the single chain diabody configurationcomprising the VL and VH of the first binding moiety and the secondbinding moiety.

TABLE C Exemplary Spacers between a Release Segment anda Bispecific Antibody Construct Amino Acid Sequence SEQ ID NO: STEPS 89SATPESGPGT 90 ATSGSETPGT 91 GTAEAASASG 92 STEPSEGSAPGTS 93 SGPGTS 94GTSTEPS 95

In some embodiments of the polypeptides of this disclosure, a releasesegment (RS) (e.g., a first release segment (RS1), a second releasesegment (RS2), etc.) can be fused to a bispecific antibody construct(BsAb) by a spacer. Such spacer can (each independently) comprise atleast 4 types of amino acids that are glycine (G), alanine (A), serine(S), threonine (T), glutamate (E) or proline (P). The peptides of thisdisclosure can comprise a first release segment fused to the bispecificantibody construct via a first spacer and a second release segment fusedto the bispecific antibody construct via a second space. A spacer (e.g.,a first spacer, a second spacer, etc.) can (each independently) comprisean amino acid sequence having at least (about) 80%, at least (about)90%, or 100% sequence identity to a sequence set forth in Table C. Thespacer (e.g., the first spacer, the second spacer, etc.) can (eachindependently) comprise an amino acid sequence identical to a sequenceset forth in Table C.

Unstructured Conformation

Typically, the XTEN component of the fusion proteins are designed tobehave like denatured peptide sequences under physiological conditions,despite the extended length of the polymer. Denatured describes thestate of a peptide in solution that is characterized by a largeconformational freedom of the peptide backbone. Most peptides andproteins adopt a denatured conformation in the presence of highconcentrations of denaturants or at elevated temperature. Peptides indenatured conformation have, for example, characteristic circulardichroism (CD) spectra and are characterized by a lack of long-rangeinteractions as determined by NMR. “Denatured conformation” and“unstructured conformation” are used synonymously herein. In some cases,the invention provides XTEN sequences that, under physiologicconditions, can resemble denatured sequences largely devoid in secondarystructure. In other cases, the XTEN sequences can be substantiallydevoid of secondary structure under physiologic conditions. “Largelydevoid,” as used in this context, means that less than 50% of the XTENamino acid residues of the XTEN sequence contribute to secondarystructure as measured or determined by the means described herein.“Substantially devoid,” as used in this context, means that at leastabout 60%, or about 70%, or about 80%, or about 90%, or about 95%, or atleast about 99% of the XTEN amino acid residues of the XTEN sequence donot contribute to secondary structure, as measured or determined by themeans described herein.

A variety of methods have been established in the art to discern thepresence or absence of secondary and tertiary structures in a givenpolypeptide. In particular, XTEN secondary structure can be measuredspectrophotometrically, e.g., by circular dichroism spectroscopy in the“far-UV” spectral region (190-250 nm). Secondary structure elements,such as alpha-helix and beta-sheet, each give rise to a characteristicshape and magnitude of CD spectra. Secondary structure can also bepredicted for a polypeptide sequence via certain computer programs oralgorithms, such as the well-known Chou-Fasman algorithm (Chou, P. Y.,et al. (1974) Biochemistry, 13: 222-45) and theGarnier-Osguthorpe-Robson (“GOR”) algorithm (Gamier J, Gibrat J F,Robson B. (1996), GOR method for predicting protein secondary structurefrom amino acid sequence. Methods Enzymol 266:540-553), as described inUS Patent Application Publication No. 20030228309A1. For a givensequence, the algorithms can predict whether there exists some or nosecondary structure at all, expressed as the total and/or percentage ofresidues of the sequence that form, for example, alpha-helices orbeta-sheets or the percentage of residues of the sequence predicted toresult in random coil formation (which lacks secondary structure).

In some cases, the XTEN sequences used in the inventive fusion proteincompositions can have an alpha-helix percentage ranging from 0% to lessthan about 5% as determined by a Chou-Fasman algorithm. In other cases,the XTEN sequences of the fusion protein compositions can have abeta-sheet percentage ranging from 0% to less than about 5% asdetermined by a Chou-Fasman algorithm. In some cases, the XTEN sequencesof the fusion protein compositions can have an alpha-helix percentageranging from 0% to less than about 5% and a beta-sheet percentageranging from 0% to less than about 5% as determined by a Chou-Fasmanalgorithm. In preferred embodiments, the XTEN sequences of the fusionprotein compositions will have an alpha-helix percentage less than about2% and a beta-sheet percentage less than about 2%. In other cases, theXTEN sequences of the fusion protein compositions can have a high degreeof random coil percentage, as determined by a GOR algorithm. In someembodiments, an XTEN sequence can have at least about 80%, morepreferably at least about 90%, more preferably at least about 91%, morepreferably at least about 92%, more preferably at least about 93%, morepreferably at least about 94%, more preferably at least about 95%, morepreferably at least about 96%, more preferably at least about 97%, morepreferably at least about 98%, and most preferably at least about 99%random coil, as determined by a GOR algorithm.

Net Charge

In other cases, the XTEN polypeptides can have an unstructuredcharacteristic imparted by incorporation of amino acid residues with anet charge and/or reducing the proportion of hydrophobic amino acids inthe XTEN sequence. The overall net charge and net charge density may becontrolled by modifying the content of charged amino acids in the XTENsequences. In some cases, the net charge density of the XTEN of thecompositions may be above +0.1 or below −0.1 charges/residue. In othercases, the net charge of a XTEN can be about 0%, about 1%, about 2%,about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%,about 10% about 11%, about 12%, about 13%, about 14%, about 15%, about16%, about 17%, about 18%, about 19%, or about 20% or more.

Since most tissues and surfaces in a human or animal have a net negativecharge, the XTEN sequences can be designed to have a net negative chargeto minimize non-specific interactions between the XTEN containingcompositions and various surfaces such as blood vessels, healthytissues, or various receptors. Not to be bound by a particular theory,the XTEN can adopt open conformations due to electrostatic repulsionbetween individual amino acids of the XTEN polypeptide that individuallycarry a high net negative charge and that are distributed across thesequence of the XTEN polypeptide. Such a distribution of net negativecharge in the extended sequence lengths of XTEN can lead to anunstructured conformation that, in turn, can result in an effectiveincrease in hydrodynamic radius. Accordingly, in one embodiment theinvention provides XTEN in which the XTEN sequences contain about 8, 10,15, 20, 25, or even about 30% glutamic acid. The XTEN of thecompositions of the present invention generally have no or a low contentof positively charged amino acids. In some cases the XTEN may have lessthan about 10% amino acid residues with a positive charge, or less thanabout 7%, or less than about 5%, or less than about 2% amino acidresidues with a positive charge. However, the invention contemplatesconstructs where a limited number of amino acids with a positive charge,such as lysine, may be incorporated into XTEN to permit conjugationbetween the epsilon amine of the lysine and a reactive group on apeptide, a linker bridge, or a reactive group on a drug or smallmolecule to be conjugated to the XTEN backbone. In the foregoing, afusion proteins can be constructed that comprises XTEN, a biologicallyactive protein, plus a chemotherapeutic agent useful in the treatment ofmetabolic diseases or disorders, wherein the maximum number of moleculesof the agent incorporated into the XTEN component is determined by thenumbers of lysines or other amino acids with reactive side chains (e.g.,cysteine) incorporated into the XTEN.

In some cases, an XTEN sequence may comprise charged residues separatedby other residues such as serine or glycine, which may lead to betterexpression or purification behavior. Based on the net charge, XTENs ofthe subject compositions may have an isoelectric point (pI) of 1.0, 1.5,2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, or even 6.5. In preferredembodiments, the XTEN will have an isoelectric point between 1.5 and4.5. In these embodiments, the XTEN incorporated into the BPXTEN fusionprotein compositions of the present invention would carry a net negativecharge under physiologic conditions that may contribute to theunstructured conformation and reduced binding of the XTEN component tomammalian proteins and tissues.

As hydrophobic amino acids can impart structure to a polypeptide, theinvention provides that the content of hydrophobic amino acids in theXTEN will typically be less than 5%, or less than 2%, or less than 10%hydrophobic amino acid content. In one embodiment, the amino acidcontent of methionine and tryptophan in the XTEN component of a BPXTENfusion protein is typically less than 5%, or less than 2%, and mostpreferably less than 1%. In some embodiments, the XTEN will have asequence that has less than 10% amino acid residues with a positivecharge, or less than about 7%, or less that about 5%, or less than about2% amino acid residues with a positive charge, the sum of methionine andtryptophan residues will be less than 2%, and the sum of asparagine andglutamine residues will be less than 10% of the total XTEN sequence.

Increased Hydrodynamic Radius

In some embodiments, the XTEN can have a high hydrodynamic radius,conferring a corresponding increased Apparent Molecular Weight to theBPXTEN fusion protein which incorporates the XTEN. The linking of XTENto BP sequences can result in BPXTEN compositions that can haveincreased hydrodynamic radii, increased Apparent Molecular Weight, andincreased Apparent Molecular Weight Factor compared to a BP not linkedto an XTEN. For example, in therapeutic applications in which prolongedhalf-life is desired, compositions in which a XTEN with a highhydrodynamic radius is incorporated into a fusion protein comprising oneor more BP can effectively enlarge the hydrodynamic radius of thecomposition beyond the glomerular pore size of approximately 3-5 nm(corresponding to an apparent molecular weight of about 70 kDa)(Caliceti. 2003. Pharmacokinetic and biodistribution properties ofpoly(ethylene glycol)-protein conjugates. Adv. Drug Deliv. Rev.55:1261-1277), resulting in reduced renal clearance of circulatingproteins. The hydrodynamic radius of a protein is determined by itsmolecular weight as well as by its structure, including shape andcompactness. Not to be bound by a particular theory, the XTEN can adoptopen conformations due to electrostatic repulsion between individualcharges of the peptide or the inherent flexibility imparted by theparticular amino acids in the sequence that lack potential to confersecondary structure. The open, extended and unstructured conformation ofthe XTEN polypeptide can have a greater proportional hydrodynamic radiuscompared to polypeptides of a comparable sequence length and/ormolecular weight that have secondary and/or tertiary structure, such astypical globular proteins. Methods for determining the hydrodynamicradius are well known in the art, such as by the use of size exclusionchromatography (SEC), as described in U.S. Pat. Nos. 6,406,632 and7,294,513. The addition of increasing lengths of XTEN results inproportional increases in the parameters of hydrodynamic radius,Apparent Molecular Weight, and Apparent Molecular Weight Factor,permitting the tailoring of BPXTEN to desired characteristic cut-offApparent Molecular Weights or hydrodynamic radii. Accordingly, incertain embodiments, the BPXTEN fusion protein can be configured with anXTEN such that the fusion protein can have a hydrodynamic radius of atleast about 5 nm, or at least about 8 nm, or at least about 10 nm, or 12nm, or at least about 15 nm. In the foregoing embodiments, the largehydrodynamic radius conferred by the XTEN in an BPXTEN fusion proteincan lead to reduced renal clearance of the resulting fusion protein,leading to a corresponding increase in terminal half-life, an increasein mean residence time, and/or a decrease in renal clearance rate.

In some embodiments, an XTEN of a chosen length and sequence can beselectively incorporated into a BPXTEN to create a fusion protein thatwill have, under physiologic conditions, an Apparent Molecular Weight ofat least about 150 kDa, or at least about 300 kDa, or at least about 400kDa, or at least about 500 kDa, or at least about 600 kDa, or at leastabout 700 kDa, or at least about 800 kDa, or at least about 900 kDa, orat least about 1000 kDa, or at least about 1200 kDa, or at least about1500 kDa, or at least about 1800 kDa, or at least about 2000 kDa, or atleast about 2300 kDa or more. In some embodiments, an XTEN of a chosenlength and sequence can be selectively linked to a BP to result in aBPXTEN fusion protein that has, under physiologic conditions, anApparent Molecular Weight Factor of at least three, alternatively of atleast four, alternatively of at least five, alternatively of at leastsix, alternatively of at least seven, alternatively of at least eight,alternatively of at least nine, alternatively of at least 10,alternatively of at least 15, or an Apparent Molecular Weight Factor ofat least 20 or greater. In some embodiments, the BPXTEN fusion proteinhas, under physiologic conditions, an Apparent Molecular Weight Factorthat is about 4 to about 20, or is about 6 to about 15, or is about 8 toabout 12, or is about 9 to about 10 relative to the actual molecularweight of the fusion protein. In some embodiments, the (fusion)polypeptide exhibits an apparent molecular weight factor underphysiological conditions that is greater than about 6.

Increased Terminal Half-Life

In some embodiments, the (fusion) polypeptide has a terminal half-lifethat is at least two-fold longer, or at least three-fold longer, or atleast four-fold longer, or at least five-fold longer, compared to thebiologically active polypeptide not linked to any XTEN. In someembodiments, the (fusion) polypeptide has a terminal half-life that isat least two-fold longer compared to the biologically active polypeptidenot linked to any XTEN.

Administration of a therapeutically effective dose of any of theembodiments of BPXTEN fusion proteins described herein to a subject inneed thereof can result in a gain in time of at least two-fold, or atleast three-fold, or at least four-fold, or at least five-fold or morespent within a therapeutic window for the fusion protein compared to thecorresponding BP not linked to the XTEN of and administered at acomparable dose to a subject.

Low Immunogenicity

In some embodiments, the invention provides compositions in which theXTEN sequences have a low degree of immunogenicity or are substantiallynon-immunogenic. Several factors can contribute to the lowimmunogenicity of XTEN, e.g., the substantially non-repetitive sequence,the unstructured conformation, the high degree of solubility, the lowdegree or lack of self-aggregation, the low degree or lack ofproteolytic sites within the sequence, and the low degree or lack ofepitopes in the XTEN sequence.

One of ordinary skill in the art will understand that, in general, apolypeptide having highly repetitive short amino acid sequences (e.g.,wherein a 200 amino acid-long sequence contain on average 20 repeats ormore of a limited set of 3- or 4-mers) and/or having contiguousrepetitive amino acid residues (e.g., wherein 5- or 6-mer sequences haveidentical amino acid residues) have a tendency to aggregate or formhigher order structures or form contacts resulting in crystalline orpseudo-crystalline structures.

In some embodiments, the XTEN sequence is substantially non-repetitive,wherein (1) the XTEN sequence has no three contiguous amino acids thatare identical amino acid types, unless the amino acid is serine, inwhich case no more than three contiguous amino acids can be serineresidues; and wherein (2) the XTEN contains no 3-amino acid sequences(3-mers) that occur more than 16, more than 14, more than 12, or morethan 10 times within a 200 amino acid-long sequence of the XTEN. One ofordinary skill in the art will understand that such substantiallynon-repetitive sequences have less tendency to aggregate and, thus,enable the design of long-sequence XTENs with a relatively low frequencyof charged amino acids that would be likely to aggregate if thesequences or amino acid residues were otherwise more repetitive.

Conformational epitopes are formed by regions of the protein surfacethat are composed of multiple discontinuous amino acid sequences of theprotein antigen. The precise folding of the protein brings thesesequences into a well-defined, stable spatial configurations, orepitopes, that can be recognized as “foreign” by the host humoral immunesystem, resulting in the production of antibodies to the protein ortriggering a cell-mediated immune response. In the latter case, theimmune response to a protein in an individual is heavily influenced byT-cell epitope recognition that is a function of the peptide bindingspecificity of that individual's HLA-DR allotype. Engagement of an MHCClass II peptide complex by a cognate T-cell receptor on the surface ofthe T-cell, together with the cross-binding of certain otherco-receptors such as the CD4 molecule, can induce an activated statewithin the T-cell. Activation leads to the release of cytokines furtheractivating other lymphocytes such as B cells to produce antibodies oractivating T killer cells as a full cellular immune response.

The ability of a peptide to bind a given MHC Class II molecule forpresentation on the surface of an APC (antigen presenting cell) isdependent on a number of factors; most notably its primary sequence. Inone embodiment, a lower degree of immunogenicity may be achieved bydesigning XTEN sequences that resist antigen processing in antigenpresenting cells, and/or choosing sequences that do not bind MHCreceptors well. The invention provides BPXTEN fusion proteins withsubstantially non-repetitive XTEN polypeptides designed to reducebinding with MHC II receptors, as well as avoiding formation of epitopesfor T-cell receptor or antibody binding, resulting in a low degree ofimmunogenicity. Avoidance of immunogenicity is, in part, a direct resultof the conformational flexibility of XTEN sequences; i.e., the lack ofsecondary structure due to the selection and order of amino acidresidues. For example, of particular interest are sequences having a lowtendency to adapt compactly folded conformations in aqueous solution orunder physiologic conditions that could result in conformationalepitopes. The administration of fusion proteins comprising XTEN, usingconventional therapeutic practices and dosing, would generally notresult in the formation of neutralizing antibodies to the XTEN sequence,and may also reduce the immunogenicity of the BP fusion partner in theBPXTEN compositions.

In one embodiment, the XTEN sequences utilized in the subject fusionproteins can be substantially free of epitopes recognized by human Tcells. The elimination of such epitopes for the purpose of generatingless immunogenic proteins has been disclosed previously; see for exampleWO 98/52976, WO 02/079232, and WO 00/3317 which are incorporated byreference herein. Assays for human T cell epitopes have been described(Stickler, M., et al. (2003) J Immunol Methods, 281: 95-108). Ofparticular interest are peptide sequences that can be oligomerizedwithout generating T cell epitopes or non-human sequences. This can beachieved by testing direct repeats of these sequences for the presenceof T-cell epitopes and for the occurrence of 6 to 15-mer and, inparticular, 9-mer sequences that are not human, and then altering thedesign of the XTEN sequence to eliminate or disrupt the epitopesequence. In some cases, the XTEN sequences are substantiallynon-immunogenic by the restriction of the numbers of epitopes of theXTEN predicted to bind MHC receptors. With a reduction in the numbers ofepitopes capable of binding to MHC receptors, there is a concomitantreduction in the potential for T cell activation as well as T cellhelper function, reduced B cell activation or upregulation and reducedantibody production. The low degree of predicted T-cell epitopes can bedetermined by epitope prediction algorithms such as, e.g., TEPITOPE(Sturniolo, T., et al. (1999) Nat Biotechnol, 17: 555-61), as shown inExample 74 of International Patent Application Publication No. WO2010/144502 A2, which is incorporated by reference in its entirety. TheTEPITOPE score of a given peptide frame within a protein is the log ofthe K_(d) (dissociation constant, affinity, off-rate) of the binding ofthat peptide frame to multiple of the most common human MHC alleles, asdisclosed in Sturniolo, T. et al. (1999) Nature Biotechnology 17:555).The score ranges over at least 20 logs, from about 10 to about −10(corresponding to binding constraints of 10e¹⁰ K_(d) to 10e⁻¹⁰ K_(d)),and can be reduced by avoiding hydrophobic amino acids that can serve asanchor residues during peptide display on MHC, such as M, I, L, V, or F.In some embodiments, an XTEN component incorporated into a BPXTEN doesnot have a predicted T-cell epitope at a TEPITOPE score of about −5 orgreater, or −6 or greater, or −7 or greater, or −8 or greater, or at aTEPITOPE score of −9 or greater. As used herein, a score of “−9 orgreater” would encompass TEPITOPE scores of 10 to −9, inclusive, butwould not encompass a score of −10, as −10 is less than −9.

In some embodiments, the inventive XTEN sequences, including thoseincorporated into the subject BPXTEN fusion proteins, can be renderedsubstantially non-immunogenic by the restriction of known proteolyticsites from the sequence of the XTEN, reducing the processing of XTENinto small peptides that can bind to MHC II receptors. In someembodiments, the XTEN sequence can be rendered substantiallynon-immunogenic by the use a sequence that is substantially devoid ofsecondary structure, conferring resistance to many proteases due to thehigh entropy of the structure. Accordingly, the reduced TEPITOPE scoreand elimination of known proteolytic sites from the XTEN may render theXTEN compositions, including the XTEN of the BPXTEN fusion proteincompositions, substantially unable to be bound by mammalian receptors,including those of the immune system. In one embodiment, an XTEN of aBPXTEN fusion protein can have >100 nM K_(d) binding to a mammalianreceptor, or greater than 500 nM K_(d), or greater than 1 μM K_(d)towards a mammalian cell surface or circulating polypeptide receptor.

Additionally, the substantially non-repetitive sequence andcorresponding lack of epitopes of such embodiments of XTEN can limit theability of B cells to bind to or be activated by XTEN. While an XTEN canmake contacts with many different B cells over its extended sequence,each individual B cell may only make one or a small number of contactswith an individual XTEN. As a result, XTENs typically may have a muchlower tendency to stimulate proliferation of B cells and thus an immuneresponse. In one embodiment, the BPXTEN may have reduced immunogenicityas compared to the corresponding BP that is not fused. In oneembodiment, the administration of up to three parenteral doses of aBPXTEN to a mammal may result in detectable anti-BPXTEN IgG at a serumdilution of 1:100 but not at a dilution of 1:1000. In some embodiments,the administration of up to three parenteral doses of an BPXTEN to amammal may result in detectable anti-BP IgG at a serum dilution of 1:100but not at a dilution of 1:1000. In some embodiments, the administrationof up to three parenteral doses of an BPXTEN to a mammal may result indetectable anti-XTEN IgG at a serum dilution of 1:100 but not at adilution of 1:1000. In the foregoing embodiments, the mammal can be amouse, a rat, a rabbit, or a cynomolgus monkey.

An additional feature of certain embodiments of XTENs with substantiallynon-repetitive sequences relative to those less non-repetitive sequences(such as one having three contiguous amino acids that are identical) canbe that non-repetitive XTENs form weaker contacts with antibodies (e.g.monovalent interactions), thereby resulting in less likelihood of immuneclearance such that the BPXTEN compositions can remain in circulationfor an increased period of time.

In some embodiments, the (fusion) polypeptide is less immunogeniccompared to the biologically active polypeptide not linked to any XTEN,wherein immunogenicity is ascertained by measuring production of IgGantibodies that selectively bind to the biologically active polypeptideafter administration of comparable doses to a subject.

Spacers & BP Release Segment

In some embodiments, at least a portion of the biological activity ofthe respective BP is retained by the intact BPXTEN. In some embodiments,the BP component either becomes biologically active or has an increasein biological activity upon its release from the XTEN by cleavage of anoptional cleavage sequence incorporated within spacer sequences into theBPXTEN, as described more fully hereinbelow.

Any spacer sequence group is optional in the fusion proteins encompassedby the invention. The spacer may be provided to enhance expression ofthe fusion protein from a host cell or to decrease steric hindrance suchthat the BP component may assume its desired tertiary structure and/orinteract appropriately with its target molecule. For spacers and methodsof identifying desirable spacers, see, for example, George, et al.(2003) Protein Engineering 15:871-879, specifically incorporated byreference herein. In one embodiment, the spacer comprises one or morepeptide sequences that are between 1 to 50 amino acid residues inlength, or about 1 to 25 residues, or about 1 to 10 residues in length.Spacer sequences, exclusive of cleavage sites, can comprise any of the20 natural L amino acids, and will preferably comprise hydrophilic aminoacids that are sterically unhindered that can include, but not belimited to, glycine (G), alanine (A), serine (S), threonine (T),glutamate (E) or proline (P). In some embodiments, the spacer can bepolyglycines or polyalanines, or predominately a mixture of combinationsof glycine and alanine residues. The spacer polypeptide exclusive of acleavage sequence is largely to substantially devoid of secondarystructure. In one embodiment, one or both spacer sequences in a BPXTENfusion protein composition may each further contain a cleavage sequence,which may be identical or may be different, wherein the cleavagesequence may be acted on by a protease to release the BP from the fusionprotein.

In some cases, the incorporation of the cleavage sequence into theBPXTEN is designed to permit release of a BP that becomes active or moreactive upon its release from the XTEN. The cleavage sequences arelocated sufficiently close to the BP sequences, generally within 18, orwithin 12, or within 6, or within 2 amino acids of the BP sequenceterminus, such that any remaining residues attached to the BP aftercleavage do not appreciably interfere with the activity (e.g., such asbinding to a receptor) of the BP, yet provide sufficient access to theprotease to be able to effect cleavage of the cleavage sequence. In someembodiments, the cleavage site is a sequence that can be cleaved by aprotease endogenous to the mammalian subject such that the BPXTEN can becleaved after administration to a subject. In such cases, the BPXTEN canserve as a prodrug or a circulating depot for the BP. Examples ofcleavage sites contemplated by the invention include, but are notlimited to, a polypeptide sequence cleavable by a mammalian endogenousprotease that is FXIa, FXIIa, kallikrein, FVIIa, FIXa, FXa, FIIa(thrombin), Elastase-2, granzyme B, MMP-12, MMP-13, MMP-17 or MMP-20, orby non-mammalian proteases such as TEV, enterokinase, PreScission™protease (rhinovirus 3C protease), or sortase A. Sequences known to becleaved by the foregoing proteases are known in the art. Exemplarycleavage sequences and cut sites within the sequences are presented inTable 7a, as well as sequence variants. For example, thrombin (activatedclotting factor II) acts on the sequence LTPRSLLV (SEQ ID NO: 222)[Rawlings N. D., et al. (2008) Nucleic Acids Res., 36: D320], whichwould be cut after the arginine at position 4 in the sequence. ActiveFIIa is produced by cleavage of FII by FXa in the presence ofphospholipids and calcium and is downstream from factor IX in thecoagulation pathway. Once activated its natural role in coagulation isto cleave fibrinogen, which then in turn, begins clot formation. FIIaactivity is tightly controlled and only occurs when coagulation isnecessary for proper hemostasis. However, as coagulation is an on-goingprocess in mammals, by incorporation of the LTPRSLLV (SEQ ID NO: 222)sequence into the BPXTEN between the BP and the XTEN, the XTEN domainwould be removed from the adjoining BP concurrent with activation ofeither the extrinsic or intrinsic coagulation pathways when coagulationis required physiologically, thereby releasing BP over time. Similarly,incorporation of other sequences into BPXTEN that are acted upon byendogenous proteases would provide for sustained release of BP that may,in certain cases, provide a higher degree of activity for the BP fromthe “prodrug” form of the BPXTEN.

In some cases, only the two or three amino acids flanking both sides ofthe cut site (four to six amino acids total) would be incorporated intothe cleavage sequence. In other cases, the known cleavage sequence canhave one or more deletions or insertions or one or two or three aminoacid substitutions for any one or two or three amino acids in the knownsequence, wherein the deletions, insertions or substitutions result inreduced or enhanced susceptibility but not an absence of susceptibilityto the protease, resulting in an ability to tailor the rate of releaseof the BP from the XTEN. Exemplary substitutions are shown in Table 7a.

TABLE 7a Protease Cleavage Sequences for BP Release ExemplaryProtease Acting SEQ ID Cleavage SEQ Upon Sequence NO Sequence ID NOMinimal Cut Site* FXIa 224 KLTR↓VVGG 244 KD/FL/T/R↓VA/VE/GT/GV FXIIa 225TMTR↓IVGG NA NA Kallikrein 226 SPFR↓STGG 245 -/-/FL/RY↓SR/RT/-/- FVIIa227 LQVR↓IVGG NA NA FIXa 228 PLGR↓IVGG 247 -/-/G/R↓-/-/-/- FXa 229IEGR↓TVGG 248 IA/E/GFP/R↓STI/VFS/-/G FIIa (thrombin) 230 LTPR↓SLLV 249-/-/PLA/R↓SAG/-/-/- Elastase-2 231 LGPV↓SGVP 250 -/-/-/VIAT↓-/-/-/-Granzyme-B 232 VAGD↓SLEE 251 V/-/-/D↓-/-/-/- MMP-12 233 GPAG↓LGGA 241G/PA/-/G↓L/-/G/- (SEQ ID NO: 241) MMP-13 234 GPAG↓LRGA 242G/P/-/G↓L/-/GA/- (SEQ ID NO: 242) MMP-17 235 APLG↓LRLR 252-/PS/-/-↓LQ/-/LT/- MMP-20 236 PALP↓LVAQ NA NA TEV 237 ENLYFQ↓G 243ENLYFQ↓/GS Enterokinase 238 DDDK↓IVGG 238 DDDK↓IVGG Protease 3C 239LEVLFQ↓GP 239 LEVLFQ↓GP (PreScission ™) Sortase A 240 LPKT↓GSES 246L/P/KEAD/T↓G/-/EKS/S ↓indicates cleavage site; NA: not applicable; *thelisting of multiple amino acids before, between, or after a slashindicate alternative amino acids that can be substituted at theposition; “-” indicates that any amino acid may be substituted for thecorresponding amino acid indicated in the middle column

In some embodiments, the BPXTEN fusion protein can comprise spacersequences that can further comprise one or more cleavage sequencesconfigured to release the BP from the fusion protein when acted on by aprotease. In some embodiments, the one or more cleavage sequences can bea sequence having at least about 80% (e.g., at least about 85%, at leastabout 90%, at least about 95%, at least about 96%, at least about 97%,at least about 98%, at least about 99%, or 100%) sequence identify to asequence from Table 7a.

In some embodiments, the disclosure provides BP release segment peptides(or release segment (RS)) that are substrates for one or more mammalianproteases associated with or produced by disease tissues or cells foundin proximity to disease tissues. Such proteases can include, but not belimited to the classes of proteases such as metalloproteinases, cysteineproteases, aspartate proteases, and serine proteases, including, but notlimited to, the proteases of Table 7b. The RS are useful for, amongstother things, incorporation into the subject recombinant polypeptides,conferring a prodrug format that can be activated by the cleavage of theRS by mammalian proteases. As described herein, the RS are incorporatedinto the subject recombinant polypeptide compositions, linking theincorporated binding moieties to the XTEN (the configurations of whichare described more fully, below) such that upon cleavage of the RS byaction of the one or more proteases for which the RS are substrates, thebinding moieties and XTEN are released from the composition and thebinding moieties, no longer shielded by the XTEN, regain their fullpotential to bind their ligands. In those recombinant polypeptidecompositions comprising a first and a second antibody fragment, thecompositions are also referred to herein as activatable antibodycompositions (AAC).

TABLE 7b Proteases of Target Tissues Class of Proteases ProteaseMetalloproteinases Meprin Neprilysin (CD10) PSMA BMP-1 A disintegrin andmetalloproteinases (ADAMs) ADAM8 ADAM9 ADAM10 ADAM12 ADAM15 ADAM17(TACE) ADAM19 ADAM28 (MDC-L) ADAM with thrombospondin motifs (ADAMTS)ADAMTS1 ADAMTS4 ADAMTS5 Matrix Metalloproteinases (MMPs) MMP-1(Collagenase 1) MMP-2 (Gelatinase A) MMP-3 (ml) MMP-7 (Matrilysin 1)MMP-8 (Collagenase 2) MMP-9 (Gelatinase B) MMP-10 (Stromelysin 2)MMP-11(Stromelysin 3) MMP-12 (Macrophage elastase) MMP-13 (Collagenase3) MMP-14 (MT1-MMP) MMP-15 (MT2-MMP) MMP-19 MMP-23 (CA-MMP) MMP-24(MT5-MMP) MMP-26 (Matrilysin 2) MMP-27 (CMMP) Cysteine ProteasesLegumain Cysteine cathepsins Cathepsin B Cathepsin C Cathepsin KCathepsin L Cathepsin S Cathepsin X Aspartate Proteases Cathepsin DCathepsin E Secretase Serine Proteases Urokinase (uPA) Tissue-typeplasminogen activator (tPA) Plasmin Thrombin Prostate-specific antigen(PSA, KLK3) Human neutrophil elastase (HNE) Elastase Tryptase Type IItransmembrane serine proteases (TTSPs) DESC1 Hepsin (HPN) MatriptaseMatriptase-2 TMPRSS2 TMPRSS3 TMPRSS4 (CAP2) Fibroblast ActivationProtein (FAP) kallikrein-related peptidase (KLK family) KLK4 KLK5 KLK6KLK7 KLK8 KLK10 KLK11 KLK13 KLK14

In one embodiment, the disclosure provides activatable recombinantpolypeptides comprising a first release segment (RS1) sequence having atleast 88%, or at least 94%, or 100% sequence identity, when optimallyaligned, to a sequence identified herein by the sequences set forth inTable 8a, wherein the RS1 is a substrate for one or more mammalianproteases. In other embodiments, the disclosure provides activatablerecombinant polypeptides comprising a RS1 and a second release segment(RS2) sequence, each having at least 88%, or at least 94%, or 100%sequence identity, when optimally aligned, to a sequence identifiedherein by the sequences set forth in Table 8a, wherein the RS1 and theRS2 each are a substrate for one or more mammalian proteases. In someembodiments, disclosure provides activatable recombinant polypeptidescomprising a first RS (RS1) sequence having at least 90%, at least 93%,at least 97%, or 100% identity, when optimally aligned, to a sequenceidentified herein by the sequences set forth in Table 8b, wherein the RSis a substrate for one or more mammalian proteases. In otherembodiments, the disclosure provides activatable recombinantpolypeptides comprising a RS1 and a second release segment (RS2)sequence, each having at least 88%, or at least 94%, or 100% sequenceidentity, when optimally aligned, to a sequence identified herein by thesequences set forth in Table 8b, wherein the RS1 and the RS2 are each asubstrate for one or more mammalian proteases (e.g., at one, two, orthree cleavage sites within each release segment sequence). In theembodiments of activatable recombinant polypeptides comprising RS1 andRS2, the two release segments can be identical or the sequences can bedifferent.

The present disclosure contemplates release segments that are substratesfor one, two or three different classes of proteases that aremetalloproteinases, cysteine proteases, aspartate proteases, or seineproteases, including the proteases of Table 7b. In a particular feature,the RS serve as substrates for proteases found in close association withor are co-localized with disease tissues or cells, such as but notlimited to tumors, cancer cells, and inflammatory tissues, and uponcleavage of the RS, the binding moieties that are otherwise shielded bythe XTEN of the subject recombinant polypeptide compositions (and thushave a lower binding affinity for their respective ligands) are releasedfrom the composition and regain their full potential to bind the targetand/or effector cell ligands. In some embodiments, the RS of the subjectrecombinant polypeptide compositions comprises an amino acid sequencethat is a substrate for a cellular protease located within a targetedcell, including but not limited to the proteases of Table 7b. In anotherparticular feature of the subject recombinant polypeptide compositions,the RS that are substrates for two or three classes of proteases weredesigned with sequences that are capable of being cleaved in differentlocations of the RS sequence by the different proteases. Thus, the RSthat are substrates for two, three, or more classes of proteases havetwo, three, or a plurality of distinct cleavage sites in the RSsequence, but cleavage by a single protease nevertheless results in therelease of the binding moieties and the XTEN from the recombinantpolypeptide composition comprising the RS.

In one embodiment, the RS of the disclosure for incorporation into thesubject recombinant polypeptide compositions is a substrate for one ormore proteases including but not limited to meprin, neprilysin (CD10),PSMA, BMP-1, A disintegrin and metalloproteinases (ADAMs), ADAM8, ADAM9,ADAM10, ADAM12, ADAM15, ADAM17 (TACE), ADAM19, ADAM28 (MDC-L), ADAM withthrombospondin motifs (ADAMTS), ADAMTS1, ADAMTS4, ADAMTS5, MMP-1(collagenase 1), matrix metalloproteinase-1 (MMP-1), matrixmetalloproteinase-2 (MMP-2, gelatinase A), matrix metalloproteinase-3(MMP-3, stromelysin 1), matrix metalloproteinase-7 (MMP-7, Matrilysin1), matrix metalloproteinase-8 (MMP-8, collagenase 2), matrixmetalloproteinase-9 (MMP-9, gelatinase B), matrix metalloproteinase-10(MMP-10, stromelysin 2), matrix metalloproteinase-11 (MMP-11,stromelysin 3), matrix metalloproteinase-12 (MMP-12, macrophageelastase), matrix metalloproteinase-13 (MMP-13, collagenase 3), matrixmetalloproteinase-14 (MMP-14, MT1-MMP), matrix metalloproteinase-15(MMP-15, MT2-MMP), matrix metalloproteinase-19 (MMP-19), matrixmetalloproteinase-23 (MMP-23, CA-MMP), matrix metalloproteinase-24(MMP-24, MT5-MMP), matrix metalloproteinase-26 (MMP-26, matrilysin 2),matrix metalloproteinase-27 (MMP-27, CMMP), legumain, cathepsin B,cathepsin C, cathepsin K, cathepsin L, cathepsin S, cathepsin X,cathepsin D, cathepsin E, secretase, urokinase (uPA), tissue-typeplasminogen activator (tPA), plasmin, thrombin, prostate-specificantigen (PSA, KLK3), human neutrophil elastase (HNE), elastase,tryptase, Type II transmembrane serine proteases (TTSPs), DESC1, hepsin(HPN), matriptase, matriptase-2, TMPRSS2, TMPRSS3, TMPRSS4 (CAP2),fibroblast activation protein (FAP), kallikrein-related peptidase (KLKfamily), KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, and KLK14.In one embodiment, the RS is a substrate for ADAM17. In one embodiment,the RS is a substrate for BMP-1. In one embodiment, the RS is asubstrate for cathepsin. In one embodiment, the RS is a substrate forHtrA1. In one embodiment, the RS is a substrate for legumain. In oneembodiment, the RS is a substrate for MMP-1. In one embodiment, the RSis a substrate for MMP-2. In one embodiment, the RS is a substrate forMMP-7. In one embodiment, the RS is a substrate for MMP-9. In oneembodiment, the RS is a substrate for MMP-11. In one embodiment, the RSis a substrate for MMP-14. In one embodiment, the RS is a substrate foruPA. In one embodiment, the RS is a substrate for matriptase. In oneembodiment, the RS is a substrate for MT-SP 1. In one embodiment, the RSis a substrate for neutrophil elastase. In one embodiment, the RS is asubstrate for thrombin. In one embodiment RS is a substrate for TMPRSS3.In one embodiment, the RS is a substrate for TMPRSS4. In one embodiment,the RS of the subject recombinant polypeptide compositions is asubstrate for at least two proteases including but not limited tolegumain, MMP-1, MMP-2, MMP-7, MMP-9, MMP-11, MMP-14, uPA, andmatriptase. In some embodiments, the RS of the subject recombinantpolypeptide compositions is a substrate for legumain, MMP-1, MMP-2,MMP-7, MMP-9, MMP-11, MMP-14, uPA, and matriptase.

TABLE 8a BP Release Segment Sequences. Construct  Amino Acid SEQ Name IDSequence ID NO RSR-1517 AC1611 EAGRSANHEPLGLVAT 7001 BSRS-A1-1 AC1605ASGRSTNAGPSGLAGP 7002 BSRS-A2-1 AC1606 ASGRSTNAGPQGLAGQ 7003 BSRS-A3-1AC1607 ASGRSTNAGPPGLTGP 7004 VP-1 AC1608 ASSRGTNAGPAGLTGP 7005 RSR-1752AC1609 ASSRTTNTGPSTLTGP 7006 RSR-1512 AC1610 AAGRSDNGTPLELVAP 7007RSR-1517 AC1611 EAGRSANHEPLGLVAT 7008 VP-2 AC1612 ASGRGTNAGPAGLTGP 7009RSR-1018 AC1613 LFGRNDNHEPLELGGG 7010 RSR-1053 AC1614 TAGRSDNLEPLGLVFG7011 RSR-1059 AC1615 LDGRSDNFHPPELVAG 7012 RSR-1065 AC1616LEGRSDNEEPENLVAG 7013 RSR-1167 AC1617 LKGRSDNNAPLALVAG 7014 RSR-1201AC1618 VYSRGTNAGPHGLTGR 7015 RSR-1218 AC1619 ANSRGTNKGFAGLIGP 7016RSR-1226 AC1620 ASSRLTNEAPAGLTIP 7017 RSR-1254 AC1621 DQSRGTNAGPEGLTDP7018 RSR-1256 AC1622 ESSRGTNIGQGGLTGP 7019 RSR-1261 AC1623SSSRGTNQDPAGLTIP 7020 RSR-1293 AC1624 ASSRGQNHSPMGLTGP 7021 RSR-1309AC1625 AYSRGPNAGPAGLEGR 7022 RSR-1326 AC1626 ASERGNNAGPANLTGF 7023RSR-1345 AC1627 ASHRGTNPKPAILTGP 7024 RSR-1354 AC1628 MSSRRTNANPAQLTGP7025 RSR-1426 AC1629 GAGRTDNHEPLELGAA 7026 RSR-1478 AC1630LAGRSENTAPLELTAG 7027 RSR-1479 AC1631 LEGRPDNHEPLALVAS 7028 RSR-1496AC1632 LSGRSDNEEPLALPAG 7029 RSR-1508 AC1633 EAGRTDNHEPLELSAP 7030RSR-1513 AC1634 EGGRSDNHGPLELVSG 7031 RSR-1516 AC1635 LSGRSDNEAPLELEAG7032 RSR-1524 AC1636 LGGRADNHEPPELGAG 7033 RSR-1622 AC1637PPSRGTNAEPAGLTGE 7034 RSR-1629 AC1638 ASTRGENAGPAGLEAP 7035 RSR-1664AC1639 ESSRGTNGAPEGLTGP 7036 RSR-1667 AC1640 ASSRATNESPAGLTGE 7037RSR-1709 AC1641 ASSRGENPPPGGLTGP 7038 RSR-1712 AC1642 AASRGTNTGPAELTGS7039 RSR-1727 AC1643 AGSRTTNAGPGGLEGP 7040 RSR-1754 AC1644APSRGENAGPATLTGA 7041 RSR-1819 AC1645 ESGRAANTGPPTLTAP 7042 RSR-1832AC1646 NPGRAANEGPPGLPGS 7043 RSR-1855 AC1647 ESSRAANLTPPELTGP 7044RSR-1911 AC1648 ASGRAANETPPGLTGA 7045 RSR-1929 AC1649 NSGRGENLGAPGLTGT7046 RSR-1951 AC1650 TTGRAANLTPAGLTGP 7047 RSR-2295 AC1761EAGRSANHTPAGLTGP 7048 RSR-2298 AC1762 ESGRAANTTPAGLTGP 7049 RSR-2038AC1679 TTGRATEAANLTPAGLTGP 7050 RSR-2072 AC1680 TTGRAEEAANLTPAGLTGP 7051RSR-2089 AC1681 TTGRAGEAANLTPAGLTGP 7052 RSR-2302 AC1682TTGRATEAANATPAGLTGP 7053 RSR-3047 AC1697 TTGRAGEAEGATSAGATGP 7054RSR-3052 AC1698 TTGEAGEAANATSAGATGP 7055 RSR-3043 AC1699TTGEAGEAAGLTPAGLTGP 7056 RSR-3041 AC1700 TTGAAGEAANATPAGLTGP 7057RSR-3044 AC1701 TTGRAGEAAGLTPAGLTGP 7058 RSR-3057 AC1702TTGRAGEAANATSAGATGP 7059 RSR-3058 AC1703 TTGEAGEAAGATSAGATGP 7060RSR-2485 AC1763 ESGRAANTEPPELGAG 7061 RSR-2486 AC1764 ESGRAANTAPEGLTGP7062 RSR-2488 AC1688 EPGRAANHEPSGLTEG 7063 RSR-2599 AC1706ESGRAANHTGAPPGGLTGP 7064 RSR-2706 AC1716 TTGRTGEGANATPGGLTGP 7065RSR-2707 AC1717 RTGRSGEAANETPEGLEGP 7066 RSR-2708 AC1718RTGRTGESANETPAGLGGP 7067 RSR-2709 AC1719 STGRTGEPANETPAGLSGP 7068RSR-2710 AC1720 TTGRAGEPANATPTGLSGP 7069 RSR-2711 AC1721RTGRPGEGANATPTGLPGP 7070 RSR-2712 AC1722 RTGRGGEAANATPSGLGGP 7071RSR-2713 AC1723 STGRSGESANATPGGLGGP 7072 RSR-2714 AC1724RTGRTGEEANATPAGLPGP 7073 RSR-2715 AC1725 ATGRPGEPANTTPEGLEGP 7074RSR-2716 AC1726 STGRSGEPANATPGGLTGP 7075 RSR-2717 AC1727PTGRGGEGANTTPTGLPGP 7076 RSR-2718 AC1728 PTGRSGEGANATPSGLTGP 7077RSR-2719 AC1729 TTGRASEGANSTPAPLTEP 7078 RSR-2720 AC1730TYGRAAEAANTTPAGLTAP 7079 RSR-2721 AC1731 TTGRATEGANATPAELTEP 7080RSR-2722 AC1732 TVGRASEEANTTPASLTGP 7081 RSR-2723 AC1733TTGRAPEAANATPAPLTGP 7082 RSR-2724 AC1734 TWGRATEPANATPAPLTSP 7083RSR-2725 AC1735 TVGRASESANATPAELTSP 7084 RSR-2726 AC1736TVGRAPEGANSTPAGLTGP 7085 RSR-2727 AC1737 TWGRATEAPNLEPATLTTP 7086RSR-2728 AC1738 TTGRATEAPNLTPAPLTEP 7087 RSR-2729 AC1739TQGRATEAPNLSPAALTSP 7088 RSR-2730 AC1740 TQGRAAEAPNLTPATLTAP 7089RSR-2731 AC1741 TSGRAPEATNLAPAPLTGP 7090 RSR-2732 AC1742TQGRAAEAANLTPAGLTEP 7091 RSR-2733 AC1743 TTGRAGSAPNLPPTGLTTP 7092RSR-2734 AC1744 TTGRAGGAENLPPEGLTAP 7093 RSR-2735 AC1745TTSRAGTATNLTPEGLTAP 7094 RSR-2736 AC1746 TTGRAGTATNLPPSGLTTP 7095RSR-2737 AC1747 TTARAGEAENLSPSGLTAP 7096 RSR-2738 AC1748TTGRAGGAGNLAPGGLTEP 7097 RSR-2739 AC1749 TTGRAGTATNLPPEGLTGP 7098RSR-2740 AC1750 TTGRAGGAANLAPTGLTEP 7099 RSR-2741 AC1751TTGRAGTAENLAPSGLTTP 7100 RSR-2742 AC1752 TTGRAGSATNLGPGGLTGP 7101RSR-2743 AC1753 TTARAGGAENLTPAGLTEP 7102 RSR-2744 AC1754TTARAGSAENLSPSGLTGP 7103 RSR-2745 AC1755 TTARAGGAGNLAPEGLTTP 7104RSR-2746 AC1756 TTSRAGAAENLTPTGLTGP 7105 RSR-2747 AC1757TYGRTTTPGNEPPASLEAE 7106 RSR-2748 AC1758 TYSRGESGPNEPPPGLTGP 7107RSR-2749 AC1759 AWGRTGASENETPAPLGGE 7108 RSR-2750 AC1760RWGRAETTPNTPPEGLETE 7109 RSR-2751 AC1765 ESGRAANHTGAEPPELGAG 7110RSR-2754 AC1801 TTGRAGEAANLTPAGLTES 7111 RSR-2755 AC1802TTGRAGEAANLTPAALTES 7112 RSR-2756 AC1803 TTGRAGEAANLTPAPLTES 7113RSR-2757 AC1804 TTGRAGEAANLTPEPLTES 7114 RSR-2758 AC1805TTGRAGEAANLTPAGLTGA 7115 RSR-2759 AC1806 TTGRAGEAANLTPEGLTGA 7116RSR-2760 AC1807 TTGRAGEAANLTPEPLTGA 7117 RSR-2761 AC1808TTGRAGEAANLTPAGLTEA 7118 RSR-2762 AC1809 TTGRAGEAANLTPEGLTEA 7119RSR-2763 AC1810 TTGRAGEAANLTPAPLTEA 7120 RSR-2764 AC1811TTGRAGEAANLTPEPLTEA 7121 RSR-2765 AC1812 TTGRAGEAANLTPEPLTGP 7122RSR-2766 AC1813 TTGRAGEAANLTPAGLTGG 7123 RSR-2767 AC1814TTGRAGEAANLTPEGLTGG 7124 RSR-2768 AC1815 TTGRAGEAANLTPEALTGG 7125RSR-2769 AC1816 TTGRAGEAANLTPEPLTGG 7126 RSR-2770 AC1817TTGRAGEAANLTPAGLTEG 7127 RSR-2771 AC1818 TTGRAGEAANLTPEGLTEG 7128RSR-2772 AC1819 TTGRAGEAANLTPAPLTEG 7129 RSR-2773 AC1820TTGRAGEAANLTPEPLTEG 7130

TABLE 8b Release Segment Sequences Amino SEQ ID Amino SEQ ID NameAcid Sequence NO: Name Acid Sequence NO: RSN-0001 GSAPGSAGGYAELRMG 7131RSC-0001 GTAEAASASGGSAGGY 7379 GAIATSGSETPGT AELRMGGAIPGSP RSN-0002GSAPGTGGGYAPLRMG 7132 RSC-0002 GTAEAASASGGTGGGY 7380 GGAATSGSETPGTAPLRMGGGAPGSP RSN-0003 GSAPGAEGGYAALRMG 7133 RSC-0003 GTAEAASASGGAEGGY7381 GEIATSGSETPGT AALRMGGEIPGSP RSN-0004 GSAPGGPGGYALLRMG 7134 RSC-0004GTAEAASASGGGPGGY 7382 GPAATSGSETPGT ALLRMGGPAPGSP RSN-0005GSAPGEAGGYAFLRMG 7135 RSC-0005 GTAEAASASGGEAGGY 7383 GSIATSGSETPGTAFLRMGGSIPGSP RSN-0006 GSAPGPGGGYASLRMG 7136 RSC-0006 GTAEAASASGGPGGGY7384 GTAATSGSETPGT ASLRMGGTAPGSP RSN-0007 GSAPGSEGGYATLRMG 7137 RSC-0007GTAEAASASGGSEGGY 7385 GAIATSGSETPGT ATLRMGGAIPGSP RSN-0008GSAPGTPGGYANLRMG 7138 RSC-0008 GTAEAASASGGTPGGY 7386 GGAATSGSETPGTANLRMGGGAPGSP RSN-0009 GSAPGASGGYAHLRMG 7139 RSC-0009 GTAEAASASGGASGGY7387 GEIATSGSETPGT AHLRMGGEIPGSP RSN-0010 GSAPGGTGGYGELRMG 7140 RSC-0010GTAEAASASGGGTGGY 7388 GPAATSGSETPGT GELRMGGPAPGSP RSN-0011GSAPGEAGGYPELRMG 7141 RSC-0011 GTAEAASASGGEAGGY 7389 GSIATSGSETPGTPELRMGGSIPGSP RSN-0012 GSAPGPGGGYVELRMG 7142 RSC-0012 GTAEAASASGGPGGGY7390 GTAATSGSETPGT VELRMGGTAPGSP RSN-0013 GSAPGSEGGYLELRMG 7143 RSC-0013GTAEAASASGGSEGGY 7391 GAIATSGSETPGT LELRMGGAIPGSP RSN-0014GSAPGTPGGYSELRMG 7144 RSC-0014 GTAEAASASGGTPGGY 7392 GGAATSGSETPGTSELRMGGGAPGSP RSN-0015 GSAPGASGGYTELRMG 7145 RSC-0015 GTAEAASASGGASGGY7393 GEIATSGSETPGT TELRMGGEIPGSP RSN-0016 GSAPGGTGGYQELRMG 7146 RSC-0016GTAEAASASGGGTGGY 7394 GPAATSGSETPGT QELRMGGPAPGSP RSN-0017GSAPGEAGGYEELRMG 7147 RSC-0017 GTAEAASASGGEAGGY 7395 GSIATSGSETPGTEELRMGGSIPGSP RSN-0018 GSAPGPGIGPAELRMGG 7148 RSC-0018 GTAEAASASGGPGIGPA7396 TAATSGSETPGT ELRMGGTAPGSP RSN-0019 GSAPGSEIGAAELRMG 7149 RSC-0019GTAEAASASGGSEIGAA 7397 GAIATSGSETPGT ELRMGGAIPGSP RSN-0020GSAPGTPIGSAELRMGG 7150 RSC-0020 GTAEAASASGGTPIGSA 7398 GAATSGSETPGTELRMGGGAPGSP RSN-0021 GSAPGASIGTAELRMG 7151 RSC-0021 GTAEAASASGGASIGTA7399 GEIATSGSETPGT ELRMGGEIPGSP RSN-0022 GSAPGGTIGNAELRMG 7152 RSC-0022GTAEAASASGGGTIGN 7400 GPAATSGSETPGT AELRMGGPAPGSP RSN-0023GSAPGEAIGQAELRMG 7153 RSC-0023 GTAEAASASGGEAIGQ 7401 GSIATSGSETPGTAELRMGGSIPGSP RSN-0024 GSAPGPGGPYAELRMG 7154 RSC-0024 GTAEAASASGGPGGPY7402 GTAATSGSETPGT AELRMGGTAPGSP RSN-0025 GSAPGSEGAYAELRMG 7155 RSC-0025GTAEAASASGGSEGAY 7403 GAIATSGSETPGT AELRMGGAIPGSP RSN-0026GSAPGTPGVYAELRMG 7156 RSC-0026 GTAEAASASGGTPGVY 7404 GGAATSGSETPGTAELRMGGGAPGSP RSN-0027 GSAPGASGLYAELRMG 7157 RSC-0027 GTAEAASASGGASGLY7405 GEIATSGSETPGT AELRMGGEIPGSP RSN-0028 GSAPGGTGIYAELRMG 7158 RSC-0028GTAEAASASGGGTGIY 7406 GPAATSGSETPGT AELRMGGPAPGSP RSN-0029GSAPGEAGFYAELRMG 7159 RSC-0029 GTAEAASASGGEAGFY 7407 GSIATSGSETPGTAELRMGGSIPGSP RSN-0030 GSAPGPGGYYAELRMG 7160 RSC-0030 GTAEAASASGGPGGYY7408 GTAATSGSETPGT AELRMGGTAPGSP RSN-0031 GSAPGSEGSYAELRMG 7161 RSC-0031GTAEAASASGGSEGSY 7409 GAIATSGSETPGT AELRMGGAIPGSP RSN-0032GSAPGTPGNYAELRMG 7162 RSC-0032 GTAEAASASGGTPGNY 7410 GGAATSGSETPGTAELRMGGGAPGSP RSN-0033 GSAPGASGEYAELRMG 7163 RSC-0033 GTAEAASASGGASGEY7411 GEIATSGSETPGT AELRMGGEIPGSP RSN-0034 GSAPGGTGHYAELRMG 7164 RSC-0034GTAEAASASGGGTGHY 7412 GPAATSGSETPGT AELRMGGPAPGSP RSN-0035GSAPGEAGGYAEARMG 7165 RSC-0035 GTAEAASASGGEAGGY 7413 GSIATSGSETPGTAEARMGGSIPGSP RSN-0036 GSAPGPGGGYAEVRMG 7166 RSC-0036 GTAEAASASGGPGGGY7414 GTAATSGSETPGT AEVRMGGTAPGSP RSN-0037 GSAPGSEGGYAEIRMG 7167 RSC-0037GTAEAASASGGSEGGY 7415 GAIATSGSETPGT AEIRMGGAIPGSP RSN-0038GSAPGTPGGYAEFRMG 7168 RSC-0038 GTAEAASASGGTPGGY 7416 GGAATSGSETPGTAEFRMGGGAPGSP RSN-0039 GSAPGASGGYAEYRMG 7169 RSC-0039 GTAEAASASGGASGGY7417 GEIATSGSETPGT AEYRMGGEIPGSP RSN-0040 GSAPGGTGGYAESRMG 7170 RSC-0040GTAEAASASGGGTGGY 7418 GPAATSGSETPGT AESRMGGPAPGSP RSN-0041GSAPGEAGGYAETRMG 7171 RSC-0041 GTAEAASASGGEAGGY 7419 GSIATSGSETPGTAETRMGGSIPGSP RSN-0042 GSAPGPGGGYAELAMG 7172 RSC-0042 GTAEAASASGGPGGGY7420 GTRATSGSETPGT AELAMGGTRPGSP RSN-0043 GSAPGSEGGYAELVMG 7173 RSC-0043GTAEAASASGGSEGGY 7421 GARATSGSETPGT AELVMGGARPGSP RSN-0044GSAPGTPGGYAELLMG 7174 RSC-0044 GTAEAASASGGTPGGY 7422 GGRATSGSETPGTAELLMGGGRPGSP RSN-0045 GSAPGASGGYAELIMG 7175 RSC-0045 GTAEAASASGGASGGY7423 GERATSGSETPGT AELIMGGERPGSP RSN-0046 GSAPGGTGGYAELWM 7176 RSC-0046GTAEAASASGGGTGGY 7424 GGPRATSGSETPGT AELWMGGPRPGSP RSN-0047GSAPGEAGGYAELSMG 7177 RSC-0047 GTAEAASASGGEAGGY 7425 GSRATSGSETPGTAELSMGGSRPGSP RSN-0048 GSAPGPGGGYAELTMG 7178 RSC-0048 GTAEAASASGGPGGGY7426 GTRATSGSETPGT AELTMGGTRPGSP RSN-0049 GSAPGSEGGYAELQMG 7179 RSC-0049GTAEAASASGGSEGGY 7427 GARATSGSETPGT AELQMGGARPGSP RSN-0050GSAPGTPGGYAELNMG 7180 RSC-0050 GTAEAASASGGTPGGY 7428 GGRATSGSETPGTAELNMGGGRPGSP RSN-0051 GSAPGASGGYAELEMG 7181 RSC-0051 GTAEAASASGGASGGY7429 GERATSGSETPGT AELEMGGERPGSP RSN-0052 GSAPGGTGGYAELRPG 7182 RSC-0052GTAEAASASGGGTGGY 7430 GPIATSGSETPGT AELRPGGPIPGSP RSN-0053GSAPGEAGGYAELRAG 7183 RSC-0053 GTAEAASASGGEAGGY 7431 GSAATSGSETPGTAELRAGGSAPGSP RSN-0054 GSAPGPGGGYAELRLG 7184 RSC-0054 GTAEAASASGGPGGGY7432 GTIATSGSETPGT AELRLGGTIPGSP RSN-0055 GSAPGSEGGYAELRIGG 7185RSC-0055 GTAEAASASGGSEGGY 7433 AAATSGSETPGT AELRIGGAAPGSP RSN-0056GSAPGTPGGYAELRSG 7186 RSC-0056 GTAEAASASGGTPGGY 7434 GGIATSGSETPGTAELRSGGGIPGSP RSN-0057 GSAPGASGGYAELRNG 7187 RSC-0057 GTAEAASASGGASGGY7435 GEAATSGSETPGT AELRNGGEAPGSP RSN-0058 GSAPGGTGGYAELRQG 7188 RSC-0058GTAEAASASGGGTGGY 7436 GPIATSGSETPGT AELRQGGPIPGSP RSN-0059GSAPGEAGGYAELRDG 7189 RSC-0059 GTAEAASASGGEAGGY 7437 GSAATSGSETPGTAELRDGGSAPGSP RSN-0060 GSAPGPGGGYAELREG 7190 RSC-0060 GTAEAASASGGPGGGY7438 GTIATSGSETPGT AELREGGTIPGSP RSN-0061 GSAPGSEGGYAELRHG 7191 RSC-0061GTAEAASASGGSEGGY 7439 GAAATSGSETPGT AELRHGGAAPGSP RSN-0062GSAPGTPGGYAELRMP 7192 RSC-0062 GTAEAASASGGTPGGY 7440 GGIATSGSETPGTAELRMPGGIPGSP RSN-0063 GSAPGASGGYAELRMA 7193 RSC-0063 GTAEAASASGGASGGY7441 GEAATSGSETPGT AELRMAGEAPGSP RSN-0064 GSAPGGTGGYAELRMV 7194 RSC-0064GTAEAASASGGGTGGY 7442 GPIATSGSETPGT AELRMVGPIPGSP RSN-0065GSAPGEAGGYAELRML 7195 RSC-0065 GTAEAASASGGEAGGY 7443 GSAATSGSETPGTAELRMLGSAPGSP RSN-0066 GSAPGPGGGYAELRMI 7196 RSC-0066 GTAEAASASGGPGGGY7444 GTIATSGSETPGT AELRMIGTIPGSP RSN-0067 GSAPGSEGGYAELRMY 7197 RSC-0067GTAEAASASGGSEGGY 7445 GAIATSGSETPGT AELRMYGAIPGSP RSN-0068GSAPGTPGGYAELRMS 7198 RSC-0068 GTAEAASASGGTPGGY 7446 GGAATSGSETPGTAELRMSGGAPGSP RSN-0069 GSAPGASGGYAELRMN 7199 RSC-0069 GTAEAASASGGASGGY7447 GEIATSGSETPGT AELRMNGEIPGSP RSN-0070 GSAPGGTGGYAELRMQ 7200 RSC-0070GTAEAASASGGGTGGY 7448 GPAATSGSETPGT AELRMQGPAPGSP RSN-0071GSAPGANHTPAGLTGP 7201 RSC-0071 GTAEAASASGGANHTP 7449 GARATSGSETPGTAGLTGPGARPGSP RSN-0072 GSAPGANTAPEGLTGPS 7202 RSC-0072 GTAEAASASGGANTAP7450 TRATSGSETPGT EGLTGPSTRPGSP RSN-0073 GSAPGTGAPPGGLTGPG 7203 RSC-0073GTAEAASASGGTGAPP 7451 TRATSGSETPGT GGLTGPGTRPGSP RSN-0074GSAPGANHEPSGLTEGS 7204 RSC-0074 GTAEAASASGGANHEP 7452 PRATSGSETPGTSGLTEGSPRPGSP RSN-0075 GSAPGANTEPPELGAGT 7205 RSC-0075 GTAEAASASGGANTEP7453 ERATSGSETPGT PELGAGTERPGSP RSN-0076 GSAPGASGPPPGLTGPP 7206 RSC-0076GTAEAASASGGASGPPP 7454 GRATSGSETPGT GLTGPPGRPGSP RSN-0077GSAPGASGTPAPLGGEP 7207 RSC-0077 GTAEAASASGGASGTP 7455 GRATSGSETPGTAPLGGEPGRPGSP RSN-0078 GSAPGPAGPPEGLETEA 7208 RSC-0078 GTAEAASASGGPAGPPE7456 GRATSGSETPGT GLETEAGRPGSP RSN-0079 GSAPGPTSGQGGLTGPE 7209 RSC-0079GTAEAASASGGPTSGQ 7457 SRATSGSETPGT GGLTGPESRPGSP RSN-0080GSAPGSAGGAANLVRG 7210 RSC-0080 GTAEAASASGGSAGGA 7458 GAIATSGSETPGTANLVRGGAIPGSP RSN-0081 GSAPGTGGGAAPLVRG 7211 RSC-0081 GTAEAASASGGTGGGA7459 GGAATSGSETPGT APLVRGGGAPGSP RSN-0082 GSAPGAEGGAAALVRG 7212 RSC-0082GTAEAASASGGAEGGA 7460 GEIATSGSETPGT AALVRGGEIPGSP RSN-0083GSAPGGPGGAALLVRG 7213 RSC-0083 GTAEAASASGGGPGGA 7461 GPAATSGSETPGTALLVRGGPAPGSP RSN-0084 GSAPGEAGGAAFLVRG 7214 RSC-0084 GTAEAASASGGEAGGA7462 GSIATSGSETPGT AFLVRGGSIPGSP RSN-0085 GSAPGPGGGAASLVRG 7215 RSC-0085GTAEAASASGGPGGGA 7463 GTAATSGSETPGT ASLVRGGTAPGSP RSN-0086GSAPGSEGGAATLVRG 7216 RSC-0086 GTAEAASASGGSEGGA 7464 GAIATSGSETPGTATLVRGGAIPGSP RSN-0087 GSAPGTPGGAAGLVRG 7217 RSC-0087 GTAEAASASGGTPGGA7465 GGAATSGSETPGT AGLVRGGGAPGSP RSN-0088 GSAPGASGGAADLVRG 7218 RSC-0088GTAEAASASGGASGGA 7466 GEIATSGSETPGT ADLVRGGEIPGSP RSN-0089GSAPGGTGGAGNLVRG 7219 RSC-0089 GTAEAASASGGGTGGA 7467 GPAATSGSETPGTGNLVRGGPAPGSP RSN-0090 GSAPGEAGGAPNLVRG 7220 RSC-0090 GTAEAASASGGEAGGA7468 GSIATSGSETPGT PNLVRGGSIPGSP RSN-0091 GSAPGPGGGAVNLVRG 7221 RSC-0091GTAEAASASGGPGGGA 7469 GTAATSGSETPGT VNLVRGGTAPGSP RSN-0092GSAPGSEGGALNLVRG 7222 RSC-0092 GTAEAASASGGSEGGA 7470 GAIATSGSETPGTLNLVRGGAIPGSP RSN-0093 GSAPGTPGGASNLVRG 7223 RSC-0093 GTAEAASASGGTPGGA7471 GGAATSGSETPGT SNLVRGGGAPGSP RSN-0094 GSAPGASGGATNLVRG 7224 RSC-0094GTAEAASASGGASGGA 7472 GEIATSGSETPGT TNLVRGGEIPGSP RSN-0095GSAPGGTGGAQNLVRG 7225 RSC-0095 GTAEAASASGGGTGGA 7473 GPAATSGSETPGTQNLVRGGPAPGSP RSN-0096 GSAPGEAGGAENLVRG 7226 RSC-0096 GTAEAASASGGEAGGA7474 GSIATSGSETPGT ENLVRGGSIPGSP RSN-1517 GSAPEAGRSANHEPLGL 7227RSC-1517 GTAEAASASGEAGRSA 7475 VATATSGSETPGT NHEPLGLVATPGSP BSRS-A1-2GSAPASGRSTNAGPSGL 7228 BSRS-A1-3 GTAEAASASGASGRST 7476 AGPATSGSETPGTNAGPSGLAGPPGSP BSRS-A2-2 GSAPASGRSTNAGPQG 7229 BSRS-A2-3GTAEAASASGASGRST 7477 LAGQATSGSETPGT NAGPQGLAGQPGSP BSRS-A3-2GSAPASGRSTNAGPPGL 7230 BSRS-A3-3 GTAEAASASGASGRST 7478 TGPATSGSETPGTNAGPPGLTGPPGSP VP-1 GSAPASSRGTNAGPAG 7231 VP-1 GTAEAASASGASSRGT 7479LTGPATSGSETPGT NAGPAGLTGPPGSP RSN-1752 GSAPASSRTTNTGPSTL 7232 RSC-1752GTAEAASASGASSRTTN 7480 TGPATSGSETPGT TGPSTLTGPPGSP RSN-1512GSAPAAGRSDNGTPLEL 7233 RSC-1512 GTAEAASASGAAGRSD 7481 VAPATSGSETPGTNGTPLELVAPPGSP RSN-1517 GSAPEAGRSANHEPLGL 7234 RSC-1517 GTAEAASASGEAGRSA7482 VATATSGSETPGT NHEPLGLVATPGSP VP-2 GSAPASGRGTNAGPAG 7235 VP-2GTAEAASASGASGRGT 7483 LTGPATSGSETPGT NAGPAGLTGPPGSP RSN-1018GSAPLFGRNDNHEPLEL 7236 RSC-1018 GTAEAASASGLFGRND 7484 GGGATSGSETPGTNHEPLELGGGPGSP RSN-1053 GSAPTAGRSDNLEPLGL 7237 RSC-1053 GTAEAASASGTAGRSD7485 VFGATSGSETPGT NLEPLGLVFGPGSP RSN-1059 GSAPLDGRSDNFHPPEL 7238RSC-1059 GTAEAASASGLDGRSD 7486 VAGATSGSETPGT NFHPPELVAGPGSP RSN-1065GSAPLEGRSDNEEPENL 7239 RSC-1065 GTAEAASASGLEGRSD 7487 VAGATSGSETPGTNEEPENLVAGPGSP RSN-1167 GSAPLKGRSDNNAPLA 7240 RSC-1167 GTAEAASASGLKGRSD7488 LVAGATSGSETPGT NNAPLALVAGPGSP RSN-1201 GSAPVYSRGTNAGPHG 7241RSC-1201 GTAEAASASGVYSRGT 7489 LTGRATSGSETPGT NAGPHGLTGRPGSP RSN-1218GSAPANSRGTNKGFAG 7242 RSC-1218 GTAEAASASGANSRGT 7490 LIGPATSGSETPGTNKGFAGLIGPPGSP RSN-1226 GSAPASSRLTNEAPAGL 7243 RSC-1226GTAEAASASGASSRLTN 7491 TIPATSGSETPGT EAPAGLTIPPGSP RSN-1254GSAPDQSRGTNAGPEG 7244 RSC-1254 GTAEAASASGDQSRGT 7492 LTDPATSGSETPGTNAGPEGLTDPPGSP RSN-1256 GSAPESSRGTNIGQGGL 7245 RSC-1256GTAEAASASGESSRGTN 7493 TGPATSGSETPGT IGQGGLTGPPGSP RSN-1261GSAPSSSRGTNQDPAGL 7246 RSC-1261 GTAEAASASGSSSRGTN 7494 TIPATSGSETPGTQDPAGLTIPPGSP RSN-1293 GSAPASSRGQNHSPMG 7247 RSC-1293 GTAEAASASGASSRGQ7495 LTGPATSGSETPGT NHSPMGLTGPPGSP RSN-1309 GSAPAYSRGPNAGPAG 7248RSC-1309 GTAEAASASGAYSRGP 7496 LEGRATSGSETPGT NAGPAGLEGRPGSP RSN-1326GSAPASERGNNAGPAN 7249 RSC-1326 GTAEAASASGASERGN 7497 LTGFATSGSETPGTNAGPANLTGFPGSP RSN-1345 GSAPASHRGTNPKPAIL 7250 RSC-1345 GTAEAASASGASHRGT7498 TGPATSGSETPGT NPKPAILTGPPGSP RSN-1354 GSAPMSSRRTNANPAQ 7251RSC-1354 GTAEAASASGMSSRRT 7499 LTGPATSGSETPGT NANPAQLTGPPGSP RSN-1426GSAPGAGRTDNHEPLE 7252 RSC-1426 GTAEAASASGGAGRTD 7500 LGAAATSGSETPGTNHEPLELGAAPGSP RSN-1478 GSAPLAGRSENTAPLEL 7253 RSC-1478 GTAEAASASGLAGRSE7501 TAGATSGSETPGT NTAPLELTAGPGSP RSN-1479 GSAPLEGRPDNHEPLAL 7254RSC-1479 GTAEAASASGLEGRPD 7502 VASATSGSETPGT NHEPLALVASPGSP RSN-1496GSAPLSGRSDNEEPLAL 7255 RSC-1496 GTAEAASASGLSGRSD 7503 PAGATSGSETPGTNEEPLALPAGPGSP RSN-1508 GSAPEAGRTDNHEPLEL 7256 RSC-1508 GTAEAASASGEAGRTD7504 SAPATSGSETPGT NHEPLELSAPPGSP RSN-1513 GSAPEGGRSDNHGPLEL 7257RSC-1513 GTAEAASASGEGGRSD 7505 VSGATSGSETPGT NHGPLELVSGPGSP RSN-1516GSAPLSGRSDNEAPLEL 7258 RSC-1516 GTAEAASASGLSGRSD 7506 EAGATSGSETPGTNEAPLELEAGPGSP RSN-1524 GSAPLGGRADNHEPPEL 7259 RSC-1524 GTAEAASASGLGGRAD7507 GAGATSGSETPGT NHEPPELGAGPGSP RSN-1622 GSAPPPSRGTNAEPAGL 7260RSC-1622 GTAEAASASGPPSRGTN 7508 TGEATSGSETPGT AEPAGLTGEPGSP RSN-1629GSAPASTRGENAGPAG 7261 RSC-1629 GTAEAASASGASTRGE 7509 LEAPATSGSETPGTNAGPAGLEAPPGSP RSN-1664 GSAPESSRGTNGAPEGL 7262 RSC-1664GTAEAASASGESSRGTN 7510 TGPATSGSETPGT GAPEGLTGPPGSP RSN-1667GSAPASSRATNESPAGL 7263 RSC-1667 GTAEAASASGASSRAT 7511 TGEATSGSETPGTNESPAGLTGEPGSP RSN-1709 GSAPASSRGENPPPGGL 7264 RSC-1709 GTAEAASASGASSRGE7512 TGPATSGSETPGT NPPPGGLTGPPGSP RSN-1712 GSAPAASRGTNTGPAEL 7265RSC-1712 GTAEAASASGAASRGT 7513 TGSATSGSETPGT NTGPAELTGSPGSP RSN-1727GSAPAGSRTTNAGPGG 7266 RSC-1727 GTAEAASASGAGSRTT 7514 LEGPATSGSETPGTNAGPGGLEGPPGSP RSN-1754 GSAPAPSRGENAGPATL 7267 RSC-1754 GTAEAASASGAPSRGE7515 TGAATSGSETPGT NAGPATLTGAPGSP RSN-1819 GSAPESGRAANTGPPTL 7268RSC-1819 GTAEAASASGESGRAA 7516 TAPATSGSETPGT NTGPPTLTAPPGSP RSN-1832GSAPNPGRAANEGPPG 7269 RSC-1832 GTAEAASASGNPGRAA 7517 LPGSATSGSETPGTNEGPPGLPGSPGSP RSN-1855 GSAPESSRAANLTPPEL 7270 RSC-1855 GTAEAASASGESSRAA7518 TGPATSGSETPGT NLTPPELTGPPGSP RSN-1911 GSAPASGRAANETPPGL 7271RSC-1911 GTAEAASASGASGRAA 7519 TGAATSGSETPGT NETPPGLTGAPGSP RSN-1929GSAPNSGRGENLGAPG 7272 RSC-1929 GTAEAASASGNSGRGE 7520 LTGTATSGSETPGTNLGAPGLTGTPGSP RSN-1951 GSAPTTGRAANLTPAG 7273 RSC-1951 GTAEAASASGTTGRAA7521 LTGPATSGSETPGT NLTPAGLTGPPGSP RSN-2295 GSAPEAGRSANHTPAG 7274RSC-2295 GTAEAASASGEAGRSA 7522 LTGPATSGSETPGT NHTPAGLTGPPGSP RSN-2298GSAPESGRAANTTPAGL 7275 RSC-2298 GTAEAASASGESGRAA 7523 TGPATSGSETPGTNTTPAGLTGPPGSP RSN-2038 GSAPTTGRATEAANLTP 7276 RSC-2038 GTAEAASASGTTGRAT7524 AGLTGPATSGSETPGT EAANLTPAGLTGPPGSP RSN-2072 GSAPTTGRAEEAANLTP 7277RSC-2072 GTAEAASASGTTGRAE 7525 AGLTGPATSGSETPGT EAANLTPAGLTGPPGSPRSN-2089 GSAPTTGRAGEAANLT 7278 RSC-2089 GTAEAASASGTTGRAG 7526PAGLTGPATSGSETPGT EAANLTPAGLTGPPGSP RSN-2302 GSAPTTGRATEAANAT 7279RSC-2302 GTAEAASASGTTGRAT 7527 PAGLTGPATSGSETPGT EAANATPAGLTGPPGSPRSN-3047 GSAPTTGRAGEAEGAT 7280 RSC-3047 GTAEAASASGTTGRAG 7528SAGATGPATSGSETPGT EAEGATSAGATGPPGSP RSN-3052 GSAPTTGEAGEAANAT 7281RSC-3052 GTAEAASASGTTGEAG 7529 SAGATGPATSGSETPGT EAANATSAGATGPPGSPRSN-3043 GSAPTTGEAGEAAGLTP 7282 RSC-3043 GTAEAASASGTTGEAG 7530AGLTGPATSGSETPGT EAAGLTPAGLTGPPGSP RSN-3041 GSAPTTGAAGEAANAT 7283RSC-3041 GTAEAASASGTTGAAG 7531 PAGLTGPATSGSETPGT EAANATPAGLTGPPGSPRSN-3044 GSAPTTGRAGEAAGLT 7284 RSC-3044 GTAEAASASGTTGRAG 7532PAGLTGPATSGSETPGT EAAGLTPAGLTGPPGSP RSN-3057 GSAPTTGRAGEAANAT 7285RSC-3057 GTAEAASASGTTGRAG 7533 SAGATGPATSGSETPGT EAANATSAGATGPPGSPRSN-3058 GSAPTTGEAGEAAGAT 7286 RSC-3058 GTAEAASASGTTGEAG 7534SAGATGPATSGSETPGT EAAGATSAGATGPPGSP RSN-2485 GSAPESGRAANTEPPEL 7287RSC-2485 GTAEAASASGESGRAA 7535 GAGATSGSETPGT NTEPPELGAGPGSP RSN-2486GSAPESGRAANTAPEGL 7288 RSC-2486 GTAEAASASGESGRAA 7536 TGPATSGSETPGTNTAPEGLTGPPGSP RSN-2488 GSAPEPGRAANHEPSGL 7289 RSC-2488 GTAEAASASGEPGRAA7537 TEGATSGSETPGT NHEPSGLTEGPGSP RSN-2599 GSAPESGRAANHTGAP 7290RSC-2599 GTAEAASASGESGRAA 7538 PGGLTGPATSGSETPGT NHTGAPPGGLTGPPGSPRSN-2706 GSAPTTGRTGEGANAT 7291 RSC-2706 GTAEAASASGTTGRTG 7539PGGLTGPATSGSETPGT EGANATPGGLTGPPGSP RSN-2707 GSAPRTGRSGEAANETP 7292RSC-2707 GTAEAASASGRTGRSG 7540 EGLEGPATSGSETPGT EAANETPEGLEGPPGSPRSN-2708 GSAPRTGRTGESANETP 7293 RSC-2708 GTAEAASASGRTGRTG 7541AGLGGPATSGSETPGT ESANETPAGLGGPPGSP RSN-2709 GSAPSTGRTGEPANETP 7294RSC-2709 GTAEAASASGSTGRTG 7542 AGLSGPATSGSETPGT EPANETPAGLSGPPGSPRSN-2710 GSAPTTGRAGEPANATP 7295 RSC-2710 GTAEAASASGTTGRAG 7543TGLSGPATSGSETPGT EPANATPTGLSGPPGSP RSN-2711 GSAPRTGRPGEGANAT 7296RSC-2711 GTAEAASASGRTGRPG 7544 PTGLPGPATSGSETPGT EGANATPTGLPGPPGSPRSN-2712 GSAPRTGRGGEAANAT 7297 RSC-2712 GTAEAASASGRTGRGG 7545PSGLGGPATSGSETPGT EAANATPSGLGGPPGSP RSN-2713 GSAPSTGRSGESANATP 7298RSC-2713 GTAEAASASGSTGRSGE 7546 GGLGGPATSGSETPGT SANATPGGLGGPPGSPRSN-2714 GSAPRTGRTGEEANATP 7299 RSC-2714 GTAEAASASGRTGRTG 7547AGLPGPATSGSETPGT EEANATPAGLPGPPGSP RSN-2715 GSAPATGRPGEPANTTP 7300RSC-2715 GTAEAASASGATGRPG 7548 EGLEGPATSGSETPGT EPANTTPEGLEGPPGSPRSN-2716 GSAPSTGRSGEPANATP 7301 RSC-2716 GTAEAASASGSTGRSGE 7549GGLTGPATSGSETPGT PANATPGGLTGPPGSP RSN-2717 GSAPPTGRGGEGANTTP 7302RSC-2717 GTAEAASASGPTGRGG 7550 TGLPGPATSGSETPGT EGANTTPTGLPGPPGSPRSN-2718 GSAPPTGRSGEGANATP 7303 RSC-2718 GTAEAASASGPTGRSGE 7551SGLTGPATSGSETPGT GANATPSGLTGPPGSP RSN-2719 GSAPTTGRASEGANSTP 7304RSC-2719 GTAEAASASGTTGRAS 7552 APLTEPATSGSETPGT EGANSTPAPLTEPPGSPRSN-2720 GSAPTYGRAAEAANTT 7305 RSC-2720 GTAEAASASGTYGRAA 7553PAGLTAPATSGSETPGT EAANTTPAGLTAPPGSP RSN-2721 GSAPTTGRATEGANAT 7306RSC-2721 GTAEAASASGTTGRAT 7554 PAELTEPATSGSETPGT EGANATPAELTEPPGSPRSN-2722 GSAPTVGRASEEANTTP 7307 RSC-2722 GTAEAASASGTVGRAS 7555ASLTGPATSGSETPGT EEANTTPASLTGPPGSP RSN-2723 GSAPTTGRAPEAANATP 7308RSC-2723 GTAEAASASGTTGRAP 7556 APLTGPATSGSETPGT EAANATPAPLTGPPGSPRSN-2724 GSAPTWGRATEPANAT 7309 RSC-2724 GTAEAASASGTWGRAT 7557PAPLTSPATSGSETPGT EPANATPAPLTSPPGSP RSN-2725 GSAPTVGRASESANATP 7310RSC-2725 GTAEAASASGTVGRAS 7558 AELTSPATSGSETPGT ESANATPAELTSPPGSPRSN-2726 GSAPTVGRAPEGANSTP 7311 RSC-2726 GTAEAASASGTVGRAP 7559AGLTGPATSGSETPGT EGANSTPAGLTGPPGSP RSN-2727 GSAPTWGRATEAPNLE 7312RSC-2727 GTAEAASASGTWGRAT 7560 PATLTTPATSGSETPGT EAPNLEPATLTTPPGSPRSN-2728 GSAPTTGRATEAPNLTP 7313 RSC-2728 GTAEAASASGTTGRAT 7561APLTEPATSGSETPGT EAPNLTPAPLTEPPGSP RSN-2729 GSAPTQGRATEAPNLSP 7314RSC-2729 GTAEAASASGTQGRAT 7562 AALTSPATSGSETPGT EAPNLSPAALTSPPGSPRSN-2730 GSAPTQGRAAEAPNLTP 7315 RSC-2730 GTAEAASASGTQGRAA 7563ATLTAPATSGSETPGT EAPNLTPATLTAPPGSP RSN-2731 GSAPTSGRAPEATNLAP 7316RSC-2731 GTAEAASASGTSGRAPE 7564 APLTGPATSGSETPGT ATNLAPAPLTGPPGSPRSN-2732 GSAPTQGRAAEAANLT 7317 RSC-2732 GTAEAASASGTQGRAA 7565PAGLTEPATSGSETPGT EAANLTPAGLTEPPGSP RSN-2733 GSAPTTGRAGSAPNLPP 7318RSC-2733 GTAEAASASGTTGRAG 7566 TGLTTPATSGSETPGT SAPNLPPTGLTTPPGSPRSN-2734 GSAPTTGRAGGAENLPP 7319 RSC-2734 GTAEAASASGTTGRAG 7567EGLTAPATSGSETPGT GAENLPPEGLTAPPGSP RSN-2735 GSAPTTSRAGTATNLTP 7320RSC-2735 GTAEAASASGTTSRAG 7568 EGLTAPATSGSETPGT TATNLTPEGLTAPPGSPRSN-2736 GSAPTTGRAGTATNLPP 7321 RSC-2736 GTAEAASASGTTGRAG 7569SGLTTPATSGSETPGT TATNLPPSGLTTPPGSP RSN-2737 GSAPTTARAGEAENLSP 7322RSC-2737 GTAEAASASGTTARAG 7570 SGLTAPATSGSETPGT EAENLSPSGLTAPPGSPRSN-2738 GSAPTTGRAGGAGNLA 7323 RSC-2738 GTAEAASASGTTGRAG 7571PGGLTEPATSGSETPGT GAGNLAPGGLTEPPGSP RSN-2739 GSAPTTGRAGTATNLPP 7324RSC-2739 GTAEAASASGTTGRAG 7572 EGLTGPATSGSETPGT TATNLPPEGLTGPPGSPRSN-2740 GSAPTTGRAGGAANLA 7325 RSC-2740 GTAEAASASGTTGRAG 7573PTGLTEPATSGSETPGT GAANLAPTGLTEPPGSP RSN-2741 GSAPTTGRAGTAENLA 7326RSC-2741 GTAEAASASGTTGRAG 7574 PSGLTTPATSGSETPGT TAENLAPSGLTTPPGSPRSN-2742 GSAPTTGRAGSATNLGP 7327 RSC-2742 GTAEAASASGTTGRAG 7575GGLTGPATSGSETPGT SATNLGPGGLTGPPGSP RSN-2743 GSAPTTARAGGAENLT 7328RSC-2743 GTAEAASASGTTARAG 7576 PAGLTEPATSGSETPGT GAENLTPAGLTEPPGSPRSN-2744 GSAPTTARAGSAENLSP 7329 RSC-2744 GTAEAASASGTTARAG 7577SGLTGPATSGSETPGT SAENLSPSGLTGPPGSP RSN-2745 GSAPTTARAGGAGNLA 7330RSC-2745 GTAEAASASGTTARAG 7578 PEGLTTPATSGSETPGT GAGNLAPEGLTTPPGSPRSN-2746 GSAPTTSRAGAAENLTP 7331 RSC-2746 GTAEAASASGTTSRAG 7579TGLTGPATSGSETPGT AAENLTPTGLTGPPGSP RSN-2747 GSAPTYGRTTTPGNEPP 7332RSC-2747 GTAEAASASGTYGRTT 7580 ASLEAEATSGSETPGT TPGNEPPASLEAEPGSPRSN-2748 GSAPTYSRGESGPNEPP 7333 RSC-2748 GTAEAASASGTYSRGES 7581PGLTGPATSGSETPGT GPNEPPPGLTGPPGSP RSN-2749 GSAPAWGRTGASENET 7334RSC-2749 GTAEAASASGAWGRTG 7582 PAPLGGEATSGSETPGT ASENETPAPLGGEPGSPRSN-2750 GSAPRWGRAETTPNTPP 7335 RSC-2750 GTAEAASASGRWGRAE 7583EGLETEATSGSETPGT TTPNTPPEGLETEPGSP RSN-2751 GSAPESGRAANHTGAE 7336RSC-2751 GTAEAASASGESGRAA 7584 PPELGAGATSGSETPGT NHTGAEPPELGAGPGSPRSN-2754 GSAPTTGRAGEAANLT 7337 RSC-2754 GTAEAASASGTTGRAG 7585PAGLTESATSGSETPGT EAANLTPAGLTESPGSP RSN-2755 GSAPTTGRAGEAANLT 7338RSC-2755 GTAEAASASGTTGRAG 7586 PAALTESATSGSETPGT EAANLTPAALTESPGSPRSN-2756 GSAPTTGRAGEAANLT 7339 RSC-2756 GTAEAASASGTTGRAG 7587PAPLTESATSGSETPGT EAANLTPAPLTESPGSP RSN-2757 GSAPTTGRAGEAANLT 7340RSC-2757 GTAEAASASGTTGRAG 7588 PEPLTESATSGSETPGT EAANLTPEPLTESPGSPRSN-2758 GSAPTTGRAGEAANLT 7341 RSC-2758 GTAEAASASGTTGRAG 7589PAGLTGAATSGSETPGT EAANLTPAGLTGAPGSP RSN-2759 GSAPTTGRAGEAANLT 7342RSC-2759 GTAEAASASGTTGRAG 7590 PEGLTGAATSGSETPGT EAANLTPEGLTGAPGSPRSN-2760 GSAPTTGRAGEAANLT 7343 RSC-2760 GTAEAASASGTTGRAG 7591PEPLTGAATSGSETPGT EAANLTPEPLTGAPGSP RSN-2761 GSAPTTGRAGEAANLT 7344RSC-2761 GTAEAASASGTTGRAG 7592 PAGLTEAATSGSETPGT EAANLTPAGLTEAPGSPRSN-2762 GSAPTTGRAGEAANLT 7345 RSC-2762 GTAEAASASGTTGRAG 7593PEGLTEAATSGSETPGT EAANLTPEGLTEAPGSP RSN-2763 GSAPTTGRAGEAANLT 7346RSC-2763 GTAEAASASGTTGRAG 7594 PAPLTEAATSGSETPGT EAANLTPAPLTEAPGSPRSN-2764 GSAPTTGRAGEAANLT 7347 RSC-2764 GTAEAASASGTTGRAG 7595PEPLTEAATSGSETPGT EAANLTPEPLTEAPGSP RSN-2765 GSAPTTGRAGEAANLT 7348RSC-2765 GTAEAASASGTTGRAG 7596 PEPLTGPATSGSETPGT EAANLTPEPLTGPPGSPRSN-2766 GSAPTTGRAGEAANLT 7349 RSC-2766 GTAEAASASGTTGRAG 7597PAGLTGGATSGSETPGT EAANLTPAGLTGGPGSP RSN-2767 GSAPTTGRAGEAANLT 7350RSC-2767 GTAEAASASGTTGRAG 7598 PEGLTGGATSGSETPGT EAANLTPEGLTGGPGSPRSN-2768 GSAPTTGRAGEAANLT 7351 RSC-2768 GTAEAASASGTTGRAG 7599PEALTGGATSGSETPGT EAANLTPEALTGGPGSP RSN-2769 GSAPTTGRAGEAANLT 7352RSC-2769 GTAEAASASGTTGRAG 7600 PEPLTGGATSGSETPGT EAANLTPEPLTGGPGSPRSN-2770 GSAPTTGRAGEAANLT 7353 RSC-2770 GTAEAASASGTTGRAG 7601PAGLTEGATSGSETPGT EAANLTPAGLTEGPGSP RSN-2771 GSAPTTGRAGEAANLT 7354RSC-2771 GTAEAASASGTTGRAG 7602 PEGLTEGATSGSETPGT EAANLTPEGLTEGPGSPRSN-2772 GSAPTTGRAGEAANLT 7355 RSC-2772 GTAEAASASGTTGRAG 7603PAPLTEGATSGSETPGT EAANLTPAPLTEGPGSP RSN-2773 GSAPTTGRAGEAANLT 7356RSC-2773 GTAEAASASGTTGRAG 7604 PEPLTEGATSGSETPGT EAANLTPEPLTEGPGSPRSN-3047 GSAPTTGRAGEAEGAT 7357 RSC-3047 GTAEAASASGTTGRAG 7605SAGATGPATSGSETPGT EAEGATSAGATGPPGSP RSN-2783 GSAPEAGRSAEATSAG 7358RSC-2783 GTAEAASASGEAGRSA 7606 ATGPATSGSETPGT EATSAGATGPPGSP RSN-3107GSAPSASGTYSRGESGP 7359 RSC-3107 GTAEAASASGSASGTYS 7607 GSPATSGSETPGTRGESGPGSPPGSP RSN-3103 GSAPSASGEAGRTDTHP 7360 RSC-3103 GTAEAASASGSASGEA7608 GSPATSGSETPGT GRTDTHPGSPPGSP RSN-3102 GSAPSASGEPGRAAEHP 7361RSC-3102 GTAEAASASGSASGEPG 7609 GSPATSGSETPGT RAAEHPGSPPGSP RSN-3119GSAPSPAGESSRGTTIA 7362 RSC-3119 GTAEAASASGSPAGESS 7610 GSPATSGSETPGTRGTTIAGSPPGSP RSN-3043 GSAPTTGEAGEAAGLTP 7363 RSC-3043 GTAEAASASGTTGEAG7611 AGLTGPATSGSETPGT EAAGLTPAGLTGPPGSP RSN-2789 GSAPEAGESAGATPAG 7364RSC-2789 GTAEAASASGEAGESA 7612 LTGPATSGSETPGT GATPAGLTGPPGSP RSN-3109GSAPSASGAPLELEAGP 7365 RSC-3109 GTAEAASASGSASGAPL 7613 GSPATSGSETPGTELEAGPGSPPGSP RSN-3110 GSAPSASGEPPELGAGP 7366 RSC-3110 GTAEAASASGSASGEPP7614 GSPATSGSETPGT ELGAGPGSPPGSP RSN-3111 GSAPSASGEPSGLTEGP 7367RSC-3111 GTAEAASASGSASGEPS 7615 GSPATSGSETPGT GLTEGPGSPPGSP RSN-3112GSAPSASGTPAPLTEPP 7368 RSC-3112 GTAEAASASGSASGTPA 7616 GSPATSGSETPGTPLTEPPGSPPGSP RSN-3113 GSAPSASGTPAELTEPP 7369 RSC-3113 GTAEAASASGSASGTPA7617 GSPATSGSETPGT ELTEPPGSPPGSP RSN-3114 GSAPSASGPPPGLTGPP 7370RSC-3114 GTAEAASASGSASGPPP 7618 GSPATSGSETPGT GLTGPPGSPPGSP RSN-3115GSAPSASGTPAPLGGEP 7371 RSC-3115 GTAEAASASGSASGTPA 7619 GSPATSGSETPGTPLGGEPGSPPGSP RSN-3125 GSAPSPAGAPEGLTGPA 7372 RSC-3125 GTAEAASASGSPAGAPE7620 GSPATSGSETPGT GLTGPAGSPPGSP RSN-3126 GSAPSPAGPPEGLETEA 7373RSC-3126 GTAEAASASGSPAGPPE 7621 GSPATSGSETPGT GLETEAGSPPGSP RSN-3127GSAPSPTSGQGGLTGPG 7374 RSC-3127 GTAEAASASGSPTSGQG 7622 SEPATSGSETPGTGLTGPGSEPPGSP RSN-3131 GSAPSESAPPEGLETEST 7375 RSC-3131GTAEAASASGSESAPPE 7623 EPATSGSETPGT GLETESTEPPGSP RSN-3132GSAPSEGSEPLELGAAS 7376 RSC-3132 GTAEAASASGSEGSEPL 7624 ETPATSGSETPGTELGAASETPPGSP RSN-3133 GSAPSEGSGPAGLEAPS 7377 RSC-3133 GTAEAASASGSEGSGPA7625 ETPATSGSETPGT GLEAPSETPPGSP RSN-3138 GSAPSEPTPPASLEAEPG 7378RSC-3138 GTAEAASASGSEPTPPA 7626 SPATSGSETPGT SLEAEPGSPPGSP

In some embodiments, the RS for incorporation into the subjectrecombinant polypeptides can be designed to be selectively sensitive inorder to have different rates of cleavage and different cleavageefficiencies to the various proteases for which they are substrates. Asa given protease may be found in different concentrations in diseasedtissues, including but not limited to a tumor, a blood cancer, or aninflammatory tissue or site of inflammation, compared to healthy tissuesor in the circulation, the disclosure provides RS that have had theindividual amino acid sequences engineered to have a higher or lowercleavage efficiency for a given protease in order to ensure that therecombinant polypeptide is preferentially converted from the prodrugform to the active form (i.e., by the separation and release of thebinding moieties and XTEN from the recombinant polypeptide aftercleavage of the RS) when in proximity to the target cell or tissue andits co-localized proteases compared to the rate of cleavage of the RS inhealthy tissue or the circulation such that the released antibodyfragment binding moieties have a greater ability to bind to ligands inthe diseased tissues compared to the prodrug form that remains incirculation. By such selective designs, the therapeutic index of theresulting compositions can be improved, resulting in reduced sideeffects relative to convention therapeutics that do not incorporate suchsite-specific activation.

As used herein cleavage efficiency is defined as the log₂ value of theratio of the percentage of the test substrate comprising the RS cleavedto the percentage of the control substrate AC1611 cleaved when each issubjected to the protease enzyme in biochemical assays (further detailedin the Examples) in which reaction in conducted wherein the initialsubstrate concentration is 6 μM, the reactions are incubated at 37° C.for 2 hours before being stopped by adding EDTA, with the amount ofdigestion products and uncleaved substrate analyzed by non-reducingSDS-PAGE to establish the ratio of the percentage cleaved. The cleavageefficiency is calculated as follows:

${Log}_{2}{\left( \frac{\%{Cleaved}{for}{substrate}{of}{interest}}{\%{cleaved}{for}{AC}1611{in}{the}{same}{experiment}} \right).}$

Thus, a cleavage efficiency of −1 means that the amount of testsubstrate cleaved was 50% compared to that of the control substrate,while a cleavage efficiency of +1 means that the amount of testsubstrate cleaved was 200% compared to that of the control substrate. Ahigher rate of cleavage by the test protease relative to the controlwould result in a higher cleavage efficiency, and a slower rate ofcleavage by the test protease relative to the control would result in alower cleavage efficiency. As detailed in the Examples, a control RSsequence AC1611 (RSR-1517), having the amino acid sequenceEAGRSANHEPLGLVAT (SEQ ID NO: 7001), was established as having anappropriate baseline cleavage efficiency by the proteases legumain,MMP-2, MMP-7, MMP-9, MMP-14, uPA, and matriptase, when tested in invitro biochemical assays for rates of cleavage by the individualproteases. By selective substitution of amino acids at individuallocations in the RS peptides, libraries of RS were created and evaluatedagainst the panel of the 7 proteases (detailed more fully in theExamples), resulting in profiles that were used to establish guidelinesfor appropriate amino acid substitutions in order to achieve RS withdesired cleavage efficiencies. In making RS with desired cleavageefficiencies, substitutions using the hydrophilic amino acids A, E, G,P, S, and T are preferred, however other L-amino acids can besubstituted at given positions in order to adjust the cleavageefficiency so long as the RS retains at least some susceptibility tocleavage by a protease. Conservative substitutions of amino acids in apeptide to retain or effect activity is well within the knowledge andcapabilities of a person within skill in the art. In one embodiment, thedisclosure provides RS in which the RS is cleaved by a proteaseincluding but not limited to legumain, MMP-1, MMP-2, MMP-7, MMP-9,MMP-11, MMP-14, uPA, or matriptase with at least a 0.2 log 2, or 0.4 log2, or 0.8 log 2, or 1.0 log₂ higher cleavage efficiency in an in vitrobiochemical competitive assay compared to the cleavage by the sameprotease of a control sequence RSR-1517 having the sequenceEAGRSANHEPLGLVAT (SEQ ID NO. 7001). In some embodiments, the disclosureprovides RS in which the RS is cleaved by a protease including but notlimited to legumain, MMP-1, MMP-2, MMP-7, MMP-9, MMP-11, MMP-14, uPA, ormatriptase with at least a 0.2 log₂, or 0.4 log₂, or 0.8 log 2, or 1.0log₂ lower cleavage efficiency in an in vitro biochemical competitiveassay compared to the cleavage by the same protease of a controlsequence RSR-1517 having the sequence EAGRSANHEPLGLVAT (SEQ ID NO.7001). In one embodiment, the disclosure provides RS in which the rateof cleavage of the RS by a protease including but not limited tolegumain, MMP-1, MMP-2, MMP-7, MMP-9, MMP-11, MMP-14, uPA, or matriptaseis at least 2-fold, or at least 4-fold, or at least 8 fold, or at least16-fold faster compared to the control sequence RSR-1517 having thesequence EAGRSANHEPLGLVAT (SEQ ID NO. 7001). In some embodiments, thedisclosure provides RS in which the rate of cleavage of the RS by aprotease including but not limited to legumain, MMP-1, MMP-2, MMP-7,MMP-9, MMP-11, MMP-14, uPA, or matriptase is at least 2-fold, or atleast 4-fold, or at least 8 fold, or at least 16-fold slower compared tothe control sequence RSR-1517 having the sequence EAGRSANHEPLGLVAT (SEQID NO. 7001).

In some embodiments, the disclosure provides AAC comprising multiple RSwherein each RS sequence is identified herein by the group of sequencesset forth in Table 8a and the RS are linked to each other by 1 to 6amino acids that are glycine, serine, alanine, and threonine. In oneembodiment, the AAC comprises a first RS and a second RS different fromthe first RS wherein each RS sequence is identified herein by thesequences set forth in Table 8a and the RS are linked to each other by 1to 6 amino acids that are glycine, serine, alanine, and threonine. Insome embodiments, the AAC comprises a first RS, a second RS differentfrom the first RS, and a third RS different from the first and thesecond RS wherein each sequence is identified herein by the sequencesset forth in Table 8a and the first and the second and the third RS arelinked to each other by 1 to 6 amino acids that are glycine, serine,alanine, and threonine. It is specifically intended that the multiple RSof the AAC can be concatenated to form a sequence that can be cleaved bymultiple proteases at different rates or efficiency of cleavage. In someembodiments, the disclosure provides AAC comprising an RS1 and an RS2identified herein by the sequences set forth in Tables 8a-8b and an XTEN1 and XTEN 2, such as those described hereinabove or described elsewhereherein, wherein the RS1 is fused between the XTEN1 and the bindingmoieties and the RS2 is fused between the XTEN2 and the bindingmoieties. It is contemplated that such compositions would be morereadily cleaved by diseased target tissues that express multipleproteases, compared with healthy tissues or when in the normalcirculation, with the result that the resulting fragments bearing thebinding moieties would more readily penetrate the target tissue; e.g., atumor, and have an enhanced ability to bind and link the target cell andthe effector cell (or just the target cell in the case of AAC designedwith a single binding moiety.

The RS of the disclosure are useful for inclusion in recombinantpolypeptides as therapeutics for treatment of cancers, autoimmunediseases, inflammatory diseases and other conditions where localizedactivation of the recombinant polypeptide is desirable. The subjectcompositions address an unmet need and are superior in one or moreaspects including enhanced terminal half-life, targeted delivery, andimproved therapeutic ratio with reduced toxicity to healthy tissuescompared to conventional antibody therapeutics or bispecific antibodytherapeutics that are active upon injection.

In some embodiments, the (fusion) polypeptide comprises a first releasesegment (RS1) positioned between the (first) XTEN and the biologicallyactive polypeptide. In some embodiments, the polypeptide furthercomprises a second release segment (RS2) positioned between thebiologically active polypeptide and the second XTEN. In someembodiments, RS1 and RS2 are identical in sequence. In some embodiments,RS1 and RS2 are not identical in sequence. In some embodiments, the RS1comprises an amino acid sequence having at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequenceidentified herein by those in Tables 8a-8b or a subset thereof. In someembodiments, the RS2 comprises an amino acid sequence having at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequenceidentity to a sequence identified herein by those in Tables 8a-8b or asubset thereof. In some embodiments, the RS1 and RS2 are each asubstrate for cleavage by multiple proteases at one, two, or threecleavage sites within each release segment sequence.

Reference Fragment

In some embodiments, the (fusion) polypeptide further comprises one ormore reference fragments releasable from the polypeptide upon digestionby the protease. In some embodiments, the one or more referencefragments each comprise a portion of the biologically activepolypeptide. In some embodiments, the one or more reference fragments isa single reference fragment that differs in sequence and molecularweight from all other peptide fragments that are releasable from thepolypeptide upon digestion of the polypeptide by the protease.

Exemplary Polypeptides

In some embodiments of the compositions of this disclosure, thepolypeptide is a recombinant polypeptide comprising an amino acidsequence having at least (about) 80% sequence identity to a sequence setforth in Table D (consisting of SEQ ID NOS: 12-47) or a subset thereof.The polypeptide can comprise an amino acid sequence having at least(about) 81%, at least (about) 82%, at least (about) 83%, at least(about) 84%, at least (about) 85%, at least (about) 86%, at least(about) 87%, at least (about) 88%, at least (about) 89%, at least(about) 90%, at least (about) 91%, at least (about) 92%, at least(about) 93%, at least (about) 94%, at least (about) 95%, at least(about) 96%, at least (about) 97%, at least (about) 98%, at least(about) 99%, or (about) 100% sequence identity to a sequence set forthin Table D (SEQ ID NOS: 12-47) or a subset thereof. The polypeptide cancomprise an amino acid sequence having at least (about) 90%, at least(about) 91%, at least (about) 92%, at least (about) 93%, at least(about) 94%, at least (about) 95%, at least (about) 96%, at least(about) 97%, at least (about) 98%, at least (about) 99%, or (about) 100%sequence identity to a sequence set forth in Table D (SEQ ID NOS: 12-47)or a subset thereof. The polypeptide can comprise an amino acid sequenceidentical to a sequence set forth in Table D (SEQ ID NOS: 12-47) or asubset thereof. It is specifically contemplated that the compositions ofthis disclosure can comprise sequence variants of the amino acidsequences set forth in Table D, such as with linker sequence(s) insertedor with purification tag sequence(s) attached thereto, so long as thevariants exhibit substantially similar or same bioactivity/bioactivitiesand/or activation mechanism(s).

TABLE D Exemplary amino acid sequences of polypeptides SEQ ID NO.Amino Acid Sequences 12ASSPAGSPTSTESGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSTPAESGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGGSAPLGGRADNHEPPELGAGATSGSETPGTDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGATPPETGAETESPGETTGGSAESEPPGEGEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSGGGSELVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSSLGGSAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVLGATPPETGAETESPGETTGGSAESEPPGEGEVQLQESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSGTAEAASASGLGGRADNHEPPELGAGSAGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSTETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTATESPEGSAPGTSESATPESGPGTSTEPSEGSAPGTSAESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTESAS 13ASSPAGSPTSTESGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSTPAESGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGEEPATSGSTPEGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGGSAPASTRGENAGPAGLEAPATSGSETPGTDIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIKGATPPETGAETESPGETTGGSAESEPPGEGEVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSGGGSELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVLGATPPETGAETESPGETTGGSAESEPPGEGEVQLLESGGGIVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTVYLQMNNLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSSGTAEAASASGASTRGENAGPAGLEAPPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTESTPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGEPEA 14ASSPAGSPTSTESGTSESATPESGPGTETEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSTPAESGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGESPATSGSTPEGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGGSAPEAGRSANHTPAGLTGPATSGSETPGTEIVLTQSPATLSLSPGERATLSCKASQDVSIGVAWYQQKPGKAPRLLIYSASYRYSGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQYYIYPYTFGQGTKVEIKGATPPETGAETESPGETTGGSAESEPPGEGQVQLVQSGVEVKKPGASVKVSCKASGFTFTDYTMDWVRQAPGQGLEWMADVNPNSGGSIYNQRFKGRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARNLGPSFYFDYWGQGTLVTVSSGGGSELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSSLGGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVLGATPPETGAETESPGETTGGSAESEPPGEGEVQLQESGGGIVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTVYLQMNNLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSSGTAEAASASGEAGRSANHTPAGLTGPTPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSESATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESA 15SPAGSPTSTESGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSTPAESGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGGSAPLFGRNDNHEPLELGGGATSGSETPGTDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGATPPETGAETESPGETTGGSAESEPPGEGEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSGGGSELVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSSLGGSAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVLGATPPETGAETESPGETTGGSAESEPPGEGEVQLQESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSGTAEAASASGLFGRNDNHEPLELGGGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTESTPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPG 16ASSPAGSPTSTESGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSTPAESGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGGSAPRTGRTGESANETPAGLGGPATSGSETPGTEIVLTQSPATLSLSPGERATLSCKASQDVSIGVAWYQQKPGQAPRLLIYSASYRYSGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQYYIYPYTFGQGTKVEIKGATPPETGAETESPGETTGGSAESEPPGEGQVQLVQSGVEVKKPGASVKVSCKASGFTFTDYTMDWVRQAPGQGLEWMADVNPNSGGSIYNQRFKGRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARNLGPSFYFDYWGQGTLVTVSSGGGSELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVLGATPPETGAETESPGETTGGSAESEPPGEGEVQLLESGGGIVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTVYLQMNNLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSSGTAEAASASGRTGRTGESANETPAGLGGPGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSTETGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATS 17ASSPAGSPTSTESGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSTPAESGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGGSAPGAGRTDNHEPLELGAAATSGSETPGTDIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTYPYTFGQGTKVEIKGATPPETGAETESPGETTGGSAESEPPGEGEVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSGGGSELVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVLGATPPETGAETESPGETTGGSAESEPPGEGEVQLLESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSGTAEAASASGGAGRTDNHEPLELGAATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSESATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESA 18GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSESATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPESGRAANTGPPTLTAPATSGSETPGTDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGATPPETGAETESPGETTGGSAESEPPGEGEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSGGGSELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSSLGGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVLGATPPETGAETESPGETTGGSAESEPPGEGEVQLQESGGGIVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTVYLQMNNLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSSGTAEAASASGESGRAANTGPPTLTAPPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTESTPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPG 19ASSPAGSPTSTESGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSTPAESGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGGSAPASGRSTNAGPPGLTGPATSGSETPGTDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGATPPETGAETESPGETTGGSAESEPPGEGEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSGGGSELVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVLGATPPETGAETESPGETTGGSAESEPPGEGEVQLLESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSGTAEAASASGASGRSTNAGPPGLTGPPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTESTPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPG 20SAGSPSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSTPAESGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSTETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTESASLFGRNDNHEPLELGGGATSGSETPGTEIVLTQSPATLSLSPGERATLSCKASQDVSIGVAWYQQKPGQAPRLLIYSASYRYSGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQYYTYPYTFGQGTKVEIKGATPPETGAETESPGETTGGSAESEPPGEGQVQLVQSGVEVKKPGASVKVSCKASGFTFTDYTMDWVRQAPGQGLEWMADVNPNSGGSIYNQRFKGRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARNLGPSFYFDYWGQGTLVTVSSGGGSELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVLGATPPETGAETESPGETTGGSAESEPPGEGEVQLLESGGGIVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTVYLQMNNLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSSGTAEAASASGLFGRNDNHEPLELGGGEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESASPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESAT 21GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPATSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESASPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPTTGRAGEAEGATSAGATGPATSGSETPGTDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGATPPETGAETESPGETTGGSAESEPPGEGEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSGGGSELVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVLGATPPETGAETESPGETTGGSAESEPPGEGEVQLLESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSGTAEAASASGTTGRAGEAEGATSAGATGPGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSTETGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATS 22ASSPAGSPTSTESGTSESATPESGPGTETEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSTPAESGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGESPATSGSTPEGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGGSAPASGRGTNAGPAGLTGPATSGSETPGTDIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIKGATPPETGAETESPGETTGGSAESEPPGEGEVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSGGGSELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSSLGGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVLGATPPETGAETESPGETTGGSAESEPPGEGEVQLQESGGGIVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTVYLQMNNLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSSGTAEAASASGASGRGTNAGPAGLTGPPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTESTPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPG 23ASSPAGSPTSTESGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSTPAESGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGEEPATSGSTPEGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGGSAPGAGRTDNHEPLELGAAATSGSETPGTDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGATPPETGAETESPGETTGGSAESEPPGEGEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSGGGSELVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSSLGGSAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVLGATPPETGAETESPGETTGGSAESEPPGEGEVQLQESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSGTAEAASASGGAGRTDNHEPLELGAAPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGftabTSESATPESGPGSEPATSGPTESGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTESTPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGEPEA 24SAGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSTPAESGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSTETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTESASASGRAANETPPGLTGAATSGSETPGTEIVLTQSPATLSLSPGERATLSCKASQDVSIGVAWYQQKPGQAPRLLIYSASYRYSGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQYYTYPYTFGQGTKVEIKGATPPETGAETESPGETTGGSAESEPPGEGQVQLVQSGVEVKKPGASVKVSCKASGFTFTDYTMDWVRQAPGQGLEWMADVNPNSGGSIYNQRFKGRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARNLGPSFYFDYWGQGTLVTVSSGGGSELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVLGATPPETGAETESPGETTGGSAESEPPGEGEVQLLESGGGIVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTVYLQMNNLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSSGTAEAASASGASGRAANETPPGLTGAGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSTETGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATS 25GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPATSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESASPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPASGRSTNAGPPGLTGPATSGSETPGTEIVLTQSPATLSLSPGERATLSCKASQDVSIGVAWYQQKPGQAPRLLIYSASYRYSGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQYYIYPYTFGQGTKVEIKGATPPETGAETESPGETTGGSAESEPPGEGQVQLVQSGVEVKKPGASVKVSCKASGFTFTDYTMDWVRQAPGQGLEWMADVNPNSGGSTYNQRFKGRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARNLGPSFYFDYWGQGTLVTVSSGGGSELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSSLGGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVLGATPPETGAETESPGETTGGSAESEPPGEGEVQLQESGGGIVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTVYLQMNNLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSSGTAEAASASGASGRSTNAGPPGLTGPGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSTETGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATS 26ASSPAGSPTSTESGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSTPAESGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGEEPATSGSTPEGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGGSAPRTGRTGESANETPAGLGGPATSGSETPGTEIVLTQSPATLSLSPGERATLSCKASQDVSIGVAWYQQKPGQAPRLLIYSASYRYSGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQYYIYPYTFGQGTKVEIKGATPPETGAETESPGETTGGSAESEPPGEGQVQLVQSGVEVKKPGASVKVSCKASGFTFTDYTMDWVRQAPGQGLEWMADVNPNSGGSIYNQRFKGRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARNLGPSFYFDYWGQGTLVTVSSGGGSELVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSSLGGSAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVLGATPPETGAETESPGETTGGSAESEPPGEGEVQLQESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSGTAEAASASGRTGRTGESANETPAGLGGPPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTESTPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPG 27ASSPAGSPTSTESGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSTPAESGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGGSAPLFGRNDNHEPLELGGGATSGSETPGTDIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTYPYTFGQGTKVEIKGATPPETGAETESPGETTGGSAESEPPGEGEVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSGGGSELVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVLGATPPETGAETESPGETTGGSAESEPPGEGEVQLLESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSGTAEAASASGLFGRNDNHEPLELGGGPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGftabTSESATPESGPGSEPATSGPTESGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTESTPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGEPEA 28ASSPAGSPTSTESGTSESATPESGPGTETEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSTPAESGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGESPATSGSTPEGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGGSAPTTGRAGEAANATSAGATGPATSGSETPGTDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGATPPETGAETESPGETTGGSAESEPPGEGEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSGGGSELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVLGATPPETGAETESPGETTGGSAESEPPGEGEVQLLESGGGIVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTVYLQMNNLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSSGTAEAASASGTTGRAGEAANATSAGATGPSAGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSTETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTATESPEGSAPGTSESATPESGPGTSTEPSEGSAPGTSAESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTESAS 29SPAGSPTSTESGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSTPAESGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGGSAPTTGRATEAANATPAGLTGPATSGSETPGTDIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTYPYTFGQGTKVEIKGATPPETGAETESPGETTGGSAESEPPGEGEVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSGGGSELVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSSLGGSAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVLGATPPETGAETESPGETTGGSAESEPPGEGEVQLQESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSGTAEAASASGTTGRATEAANATPAGLTGPTPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSESATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESA 30GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSESATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPTTARAGSAENLSPSGLTGPATSGSETPGTDIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTYPYTFGQGTKVEIKGATPPETGAETESPGETTGGSAESEPPGEGEVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSGGGSELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSSLGGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVLGATPPETGAETESPGETTGGSAESEPPGEGEVQLQESGGGIVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTVYLQMNNLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSSGTAEAASASGTTARAGSAENLSPSGLTGPEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESASPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESAT 31GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSESATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPTTGRAGEAEGATSAGATGPATSGSETPGTDIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTYPYTFGQGTKVEIKGATPPETGAETESPGETTGGSAESEPPGEGEVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSGGGSELVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVLGATPPETGAETESPGETTGGSAESEPPGEGEVQLLESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSGTAEAASASGTTGRAGEAEGATSAGATGPPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTESTPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPG 32ASSPAGSPTSTESGTSESATPESGPGTETEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSTPAESGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGESPATSGSTPEGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGGSAPPTGRSGEGANATPSGLTGPATSGSETPGTDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGATPPETGAETESPGETTGGSAESEPPGEGEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSGGGSELVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVLGATPPETGAETESPGETTGGSAESEPPGEGEVQLLESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSGTAEAASASGPTGRSGEGANATPSGLTGPPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGftabTSESATPESGPGSEPATSGPTESGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTESTPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGEPEA 33ASSPAGSPTSTESGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSTPAESGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGGSAPLGGRADNHEPPELGAGATSGSETPGTDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGATPPETGAETESPGETTGGSAESEPPGEGEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSGGGSELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVLGATPPETGAETESPGETTGGSAESEPPGEGEVQLLESGGGIVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTVYLQMNNLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSSGTAEAASASGLGGRADNHEPPELGAGTPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSESATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESA 34ASSPAGSPTSTESGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSTPAESGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGGSAPEAGRSANHTPAGLTGPATSGSETPGTDIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTYPYTFGQGTKVEIKGATPPETGAETESPGETTGGSAESEPPGEGEVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSGGGGSELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVLGATPPETGAETESPGETTGGSAESEPPGEGEVQLLESGGGIVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTVYLQMNNLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSSGTAEAASASGEAGRSANHTPAGLTGPPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTESTPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGEPEA 35ASSPAGSPTSTESGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSTPAESGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGGSAPGAGRTDNHEPLELGAAATSGSETPGTEIVLTQSPATLSLSPGERATLSCKASQDVSIGVAWYQQKPGQAPRLLIYSASYRYSGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQYYTYPYTFGQGTKVEIKGATPPETGAETESPGETTGGSAESEPPGEGQVQLVQSGVEVKKPGASVKVSCKASGFTFTDYTMDWVRQAPGQGLEWMADVNPNSGGSIYNQRFKGRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARNLGPSFYFDYWGQGTLVTVSSGGGSELVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSSLGGSAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVLGATPPETGAETESPGETTGGSAESEPPGEGEVQLQESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSGTAEAASASGGAGRTDNHEPLELGAAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTESTPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGEPEA 36GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPATSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESASPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPASGRAANETPPGLTGAATSGSETPGTEIVLTQSPATLSLSPGERATLSCKASQDVSIGVAWYQQKPGQAPRLLIYSASYRYSGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQYYIYPYTFGQGTKVEIKGATPPETGAETESPGETTGGSAESEPPGEGQVQLVQSGVEVKKPGASVKVSCKASGFTFTDYTMDWVRQAPGQGLEWMADVNPNSGGSIYNQRFKGRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARNLGPSFYFDYWGQGTLVTVSSGGGSELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSSLGGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVLGATPPETGAETESPGETTGGSAESEPPGEGEVQLQESGGGIVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTVYLQMNNLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSSGTAEAASASGASGRAANETPPGLTGASAGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSTETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTATESPEGSAPGTSESATPESGPGTSTEPSEGSAPGTSAESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTESAS 37SAGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSTPAESGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSTETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTESASESGRAANTGPPTLTAPATSGSETPGTDIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTYPYTFGQGTKVEIKGATPPETGAETESPGETTGGSAESEPPGEGEVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSGGGSELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSSLGGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVLGATPPETGAETESPGETTGGSAESEPPGEGEVQLQESGGGIVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTVYLQMNNLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSSGTAEAASASGESGRAANTGPPTLTAPSAGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSTETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTATESPEGSAPGTSESATPESGPGTSTEPSEGSAPGTSAESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTESAS 38ASSPAGSPTSTESGTSESATPESGPGTETEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSTPAESGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGESPATSGSTPEGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGGSAPASGRGTNAGPAGLTGPATSGSETPGTEIVLTQSPATLSLSPGERATLSCKASQDVSIGVAWYQQKPGKAPRLLIYSASYRYSGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQYYIYPYTFGQGTKVEIKGATPPETGAETESPGETTGGSAESEPPGEGQVQLVQSGVEVKKPGASVKVSCKASGFTFTDYTMDWVRQAPGQGLEWMADVNPNSGGSIYNQRFKGRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARNLGPSFYFDYWGQGTLVTVSSGGGSELVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVLGATPPETGAETESPGETTGGSAESEPPGEGEVQLLESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSGTAEAASASGASGRGTNAGPAGLTGPEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESASPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESAT 39ASSPAGSPTSTESGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSTPAESGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGEEPATSGSTPEGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGGSAPEAGRSANHTPAGLTGPATSGSETPGTDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGATPPETGAETESPGETTGGSAESEPPGEGEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSGGGSELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVLGATPPETGAETESPGETTGGSAESEPPGEGEVQLLESGGGIVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTVYLQMNNLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSSGTAEAASASGEAGRSANHTPAGLTGPSAGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSTETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTATESPEGSAPGTSESATPESGPGTSTEPSEGSAPGTSAESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTESAS 40ASSPAGSPTSTESGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSTPAESGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGGSAPASTRGENAGPAGLEAPATSGSETPGTDIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTYPYTFGQGTKVEIKGATPPETGAETESPGETTGGSAESEPPGEGEVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSGGGSELVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSSLGGSAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVLGATPPETGAETESPGETTGGSAESEPPGEGEVQLQESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSGTAEAASASGASTRGENAGPAGLEAPPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGftabTSESATPESGPGSEPATSGPTESGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTESTPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGEPEA 41GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSESATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPPTGRSGEGANATPSGLTGPATSGSETPGTEIVLTQSPATLSLSPGERATLSCKASQDVSIGVAWYQQKPGQAPRLLIYSASYRYSGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQYYTYPYTFGQGTKVEIKGATPPETGAETESPGETTGGSAESEPPGEGQVQLVQSGVEVKKPGASVKVSCKASGFTFTDYTMDWVRQAPGQGLEWMADVNPNSGGSIYNQRFKGRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARNLGPSFYFDYWGQGTLVTVSSGGGSELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSSLGGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVLGATPPETGAETESPGETTGGSAESEPPGEGEVQLQESGGGIVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTVYLQMNNLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSSGTAEAASASGPTGRSGEGANATPSGLTGPPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTESTPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPG 42SPAGSPTSTESGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSTPAESGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGGSAPRTGRTGESANETPAGLGGPATSGSETPGTDIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIKGATPPETGAETESPGETTGGSAESEPPGEGEVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSGGGSELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVLGATPPETGAETESPGETTGGSAESEPPGEGEVQLLESGGGIVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTVYLQMNNLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSSGTAEAASASGRTGRTGESANETPAGLGGPPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTESTPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGEPEA 43ASSPAGSPTSTESGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSTPAESGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGGSAPTTARAGSAENLSPSGLTGPATSGSETPGTDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGATPPETGAETESPGETTGGSAESEPPGEGEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSGGGSELVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVLGATPPETGAETESPGETTGGSAESEPPGEGEVQLLESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSGTAEAASASGTTARAGSAENLSPSGLTGPPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTESTPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPG 44ASSPAGSPTSTESGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSTPAESGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGEEPATSGSTPEGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGGSAPLGGRADNHEPPELGAGATSGSETPGTDIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTYPYTFGQGTKVEIKGATPPETGAETESPGETTGGSAESEPPGEGEVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSGGGSELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSSLGGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVLGATPPETGAETESPGETTGGSAESEPPGEGEVQLQESGGGIVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTVYLQMNNLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSSGTAEAASASGLGGRADNHEPPELGAGTPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSESATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESA 45SPAGSPTSTESGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSTPAESGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGGSAPTTGRAGEAEGATSAGATGPATSGSETPGTDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGATPPETGAETESPGETTGGSAESEPPGEGEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSGGGSELVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSSLGGSAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVLGATPPETGAETESPGETTGGSAESEPPGEGEVQLQESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSGTAEAASASGTTGRAGEAEGATSAGATGPEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESASPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESAT 46SAGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSTPAESGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSTETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTESASTTGRAGEAANATSAGATGPATSGSETPGTDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGATPPETGAETESPGETTGGSAESEPPGEGEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSGGGSELVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVLGATPPETGAETESPGETTGGSAESEPPGEGEVQLLESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSGTAEAASASGTTGRAGEAANATSAGATGPPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGftabTSESATPESGPGSEPATSGPTESGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTESTPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGEPEA 47ASSPAGSPTSTESGTSESATPESGPGTETEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSTPAESGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGESPATSGSTPEGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGGSAPTTGRATEAANATPAGLTGPATSGSETPGTEIVLTQSPATLSLSPGERATLSCKASQDVSIGVEIKGATPPETGAETESPGETTGGSAESEPPGEGQVQLVQSGVEVKKPGASVKVSCKASGFTFTDYTMDWVVAWYQQKPGQAPRLLIYSASYRYSGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQYYIYPYTFGQGTKRQAPGQGLEWMADVNPNSGGSIYNQRFKGRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARNLGPSFYFDYWGQGTLVTVSSGGGSELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVLGATPPETGAETESPGETTGGSAESEPPGEGEVQLLESGGGIVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTVYLQMNNLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSSGTAEAASASGTTGRATEAANATPAGLTGPPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTESTPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGEPEA

Polypeptide Mixture

Disclosed herein includes a mixture comprising a plurality ofpolypeptides of varying length; the mixture comprising a first set ofpolypeptides and a second set of polypeptides. In some embodiments, eachpolypeptide of the first set of polypeptides comprises a barcodefragment that (a) is releasable from the polypeptide by digestion with aprotease and (b) has a sequence and molecular weight that differs fromthe sequence and molecular weight of all other fragments that arereleasable from the first set of polypeptides. In some embodiments, thesecond set of polypeptides lack the barcode fragment of the first set ofpolypeptides (e.g., due to truncation). In some embodiments, both thefirst set of polypeptides and the second set of polypeptides eachcomprise a reference fragment that (a) is common to the first set ofpolypeptides and the second set of polypeptides and (b) releasable bydigestion with the protease. In some embodiments, the ratio of the firstset of polypeptides to polypeptides comprising the reference fragment isgreater than 0.70. In some embodiments, the ratio of the first set ofpolypeptides to polypeptides comprising the reference fragment isgreater than 0.80, 0.90, 0.95, or 0.98. In some embodiments, thereference fragment occurs no more than once in each polypeptide of thefirst set of polypeptides and the second set of polypeptides. In someembodiments, the protease is a protease that cleaves on the C-terminalside of glutamic acid residues. In some embodiments, the protease is aGlu-C protease. In some embodiments, the protease is not trypsin. Insome embodiments, the polypeptides of varying lengths comprisepolypeptides comprising at least one extended recombinant polypeptide(XTEN), such as any described hereinabove or described anywhere elseherein. In some embodiments, the first set of polypeptides comprises afull-length polypeptide, wherein the barcode fragment is a portion ofthe full-length polypeptide. In some embodiments, the full-lengthpolypeptide is a (fusion) polypeptide, such as any described hereinaboveor described anywhere else herein. In some embodiments, the barcodefragment lacks (does not comprise) both the N-terminal amino acid andC-terminal amino acid of the full-length polypeptide. In someembodiments, the mixture of polypeptides of varying lengths differ fromone another due to N-terminal truncation, C-terminal truncation, or bothN- and C-terminal truncation of a full-length polypeptide. In someembodiments, the first set of polypeptides and the second set ofpolypeptides may differ in one or more pharmacological properties.Non-limiting exemplary properties include.

Method of Polypeptide Characterization

Disclosed herein includes a method for assessing, in a mixturecomprising polypeptides of varying length, a relative amount of a firstset of polypeptides in the mixture to a second set of polypeptides inthe mixture, wherein (1) each polypeptide of the first set ofpolypeptides shares a barcode fragment that occurs once and only once inthe polypeptide and (2) each polypeptide of the second set ofpolypeptides lacks the barcode fragment that is shared by polypeptidesof the first set, wherein individual polypeptides of both the first ofpolypeptides and the second set of polypeptides each comprises areference fragment. The method can comprise contacting the mixture witha protease to produce a plurality of proteolytic fragments that resultfrom cleavage of the first set of polypeptides and the second set ofpolypeptides, wherein the plurality of proteolytic fragments comprise aplurality of reference fragments, and a plurality of barcode fragments.The method can further comprise determining a ratio of the amount ofbarcode fragments to the amount of reference fragments, therebyassessing the relative amounts of the first set of polypeptides to thesecond set of polypeptides. In some embodiments, the barcode fragmentoccurs no more than once in each polypeptide of the first set ofpolypeptides. In some embodiments, the reference fragment occurs no morethan once in each polypeptide of the first set of polypeptides and thesecond set of polypeptides. In some embodiments, the plurality ofproteolytic fragments comprises a plurality of reference fragments, anda plurality of barcode fragments. In some embodiments, the proteasecleaves the first and second sets of polypeptides (or the polypeptidesof varying length) on the C-terminal side of glutamic acid residues thatare not followed by a proline residue. In some embodiments, the proteaseis a Glu-C protease. In some embodiments, the protease is not trypsin.In some embodiments, the step of determining a ratio of the amount ofbarcode fragments to the amount of reference fragments comprisesidentifying barcode fragments and reference fragments from the mixtureafter it has been contacted with the protease. In some embodiments, thebarcode fragments and the reference fragments are identified based ontheir respective masses. In some embodiments, the barcode fragments andthe reference fragments are identified via mass spectrometry. In someembodiments, the barcode fragments and reference fragments areidentified via liquid chromatography-mass spectrometry (LC-MS). In someembodiments, the step of determining a ratio of the barcode fragments tothe reference fragments comprises isobaric labeling. In someembodiments, the step of determining a ratio of the barcode fragments tothe reference fragments comprises spiking the mixture with one or bothof an isotope-labeled reference fragment and an isotope labeled barcodefragment. In some embodiments, the polypeptides of varying lengthscomprise polypeptides that comprise at least one extended recombinantpolypeptide (XTEN), as described hereinabove or described anywhere elseherein. In some embodiments, the XTEN is characterized in that (i) itcomprises at least 100, or at least 150 amino acids; (ii) at least 90%of the amino acid residues of the XTEN are glycine (G), alanine (A),serine (S), threonine (T), glutamate (E) or proline (P); and (iii) itcomprises at least 4 different types of amino acids that are G, A, S, T,E, or P. In some embodiments, the barcode fragment, when present, is aportion of the XTEN. In some embodiments, the mixture of polypeptides ofvarying lengths comprises a polypeptide as any described hereinabove ordescribed anywhere else herein. In some embodiments, the polypeptides ofvarying length comprise a full-length polypeptide and truncatedfragments thereof. In some embodiments, the polypeptides of varyinglength consist essentially of the full-length polypeptide and truncatedfragments thereof. In some embodiments, the mixture of polypeptides ofvarying lengths differ from one another due to N-terminal truncation,C-terminal truncation, or both N- and C-terminal truncation of afull-length polypeptide. In some embodiments, the full-lengthpolypeptide is a polypeptide as described hereinabove or describedanywhere else herein. In some embodiments, the ratio of the amount ofbarcode fragments to reference fragments is greater than 0.50, 0.60,0.70, 0.80, 0.90, 0.95, 0.98, or 0.99.

Isobaric Labeling-Based Quantification of Peptides

In some embodiments, isobaric labeling can be used for determining aratio of the barcode fragments to the reference fragments. One ofordinary skill will understand that isobaric labeling is a massspectrometry strategy used in quantitative proteomics, wherein peptidesor proteins (or portions thereof) are labeled with various chemicalgroups that are isobaric (identical in mass) but vary in terms ofdistribution of heavy isotopes around their structure. These tags,commonly referred to as tandem mass tags, are designed so that the masstag is cleaved at a specific linker region upon high-energycollision-induced dissociation (CID) during tandem mass spectrometry,thereby yielding reporter ions of different masses. One of ordinaryskill will understand that one of the most common isobaric tags areamine-reactive tags.

The enhanced ability to detect and quantify truncation products (e.g.,via isobaric labeling) can generate knowledge than can aid in designingmanufacturing processes to include purification steps to minimize thepresence of unwanted variants in the purified drug substance/product.

Recombinant Production

The disclosure herein includes a nucleic acid. The nucleic acid cancomprise a polynucleotide (or polynucleotide sequence) encoding a(fusion) polypeptide, such as any described hereinabove or describedanywhere else herein; or the nucleic acid can comprise the reversecomplement of such a polynucleotide (or polynucleotide sequence).

The disclosure herein includes an expression vector that comprises apolynucleotide sequence, such as any described in the precedingparagraph, and a regulatory sequence operably linked to thepolynucleotide sequence.

The disclosure herein includes a host cell comprising an expressionvector, such as described any in the preceding paragraph. In someembodiments, the host cell is a prokaryote. In some embodiments, thehost cell is E. coli. In some embodiments, the host cell is a mammaliancell.

In some embodiments, the disclosure provides methods of manufacturingthe subject compositions. In one embodiment, the method comprisesculturing a host cell comprising a nucleic acid construct that encodes apolypeptide or an XTEN-containing composition of any of the embodimentsdescribed herein under conditions that promote the expression of thepolypeptide or BPXTEN fusion polypeptide, followed by recovery of thepolypeptide or BPXTEN fusion polypeptide using standard purificationmethods (e.g., column chromatography, HPLC, and the like) wherein thecomposition is recovered wherein at least 70%, or at least 80%, or atleast 90%, or at least 95%, or at least 97%, or at least 99% of thebinding fragments of the expressed polypeptide or BPXTEN fusionpolypeptide are correctly folded. In some embodiments of the method ofmaking, the expressed polypeptide or BPXTEN fusion polypeptide isrecovered in which at least or at least 90%, or at least 95%, or atleast 97%, or at least 99% of the polypeptide or BPXTEN fusionpolypeptide is recovered in monomeric, soluble form.

In some embodiments, the disclosure relates to methods of making thepolypeptide and BPXTEN fusion polypeptide at high fermentationexpression levels of functional protein using an E. coli or mammalianhost cell, as well as providing expression vectors encoding theconstructs useful in methods to produce the cytotoxically activepolypeptide construct compositions at high expression levels. In oneembodiment, the method comprises the steps of 1) preparing thepolynucleotide encoding the polypeptides of any of the embodimentsdisclosed herein, 2) cloning the polynucleotide into an expressionvector, which can be a plasmid or other vector under control ofappropriate transcription and translation sequences for high levelprotein expression in a biological system, 3) transforming anappropriate host cell with the expression vector, and 4) culturing thehost cell in conventional nutrient media under conditions suitable forthe expression of the polypeptide composition. Where desired, the hostcell is E. coli. By the method, the expression of the polypeptideresults in fermentation titers of at least 0.05 g/L, or at least 0.1g/L, or at least 0.2 g/L, or at least 0.3 g/L, or at least 0.5 g/L, orat least 0.6 g/L, or at least 0.7 g/L, or at least 0.8 g/L, or at least0.9 g/L, or at least 1 g/L, or at least 2 g/L, or at least 3 g/L, or atleast 4 g/L, or at least 5 g/L of the expression product of the hostcell and wherein at least 70%, or at least 80%, or at least 90%, or atleast 95%, or at least 97%, or at least 99% of the expressed protein arecorrectly folded. As used herein, the term “correctly folded” means thatthe antigen binding fragments component of the composition have theability to specifically bind its target ligand. In some embodiments, thedisclosure provides a method for producing a polypeptide or BPXTENfusion polypeptide, the method comprising culturing in a fermentationreaction a host cell that comprises a vector encoding a polypeptidecomprising the polypeptide or BPXTEN fusion polypeptide under conditionseffective to express the polypeptide product at a concentration of morethan about 10 milligrams/gram of dry weight host cell (mg/g), or atleast about 250 mg/g, or about 300 mg/g, or about 350 mg/g, or about 400mg/g, or about 450 mg/g, or about 500 mg/g of the polypeptide when thefermentation reaction reaches an optical density of at least 130 at awavelength of 600 nm, and wherein the antigen binding fragments of theexpressed protein are correctly folded. In some embodiments, thedisclosure provides a method for producing a polypeptide or BPXTENfusion polypeptide, the method comprising culturing in a fermentationreaction a host cell that comprises a vector encoding the compositionunder conditions effective to express the polypeptide product at aconcentration of more than about 10 milligrams/gram of dry weight hostcell (mg/g), or at least about 250 mg/g, or about 300 mg/g, or about 350mg/g, or about 400 mg/g, or about 450 mg/g, or about 500 mg/g of thepolypeptide when the fermentation reaction reaches an optical density ofat least 130 at a wavelength of 600 nm, and wherein the expressedpolypeptide product is soluble.

Pharmaceutical Composition

Disclosed herein includes a pharmaceutical composition comprising a(fusion) polypeptide, such as any described hereinabove or describedanywhere else herein, and one or more pharmaceutically acceptableexcipients. In some embodiments, the pharmaceutical composition isformulated for intradermal, subcutaneous, oral, intravenous,intra-arterial, intraabdominal, intraperitoneal, intravitreal,intrathecal, or intramuscular administration. In some embodiments, thepharmaceutical composition is in a liquid form or frozen. In someembodiments, the pharmaceutical composition is in a device that isimplanted into the eye or another body part. In some embodiments, thepharmaceutical composition is in a pre-filled syringe for a singleinjection. In some embodiments, the pharmaceutical composition isformulated as a lyophilized powder to be reconstituted prior toadministration.

In some embodiments, the dose is administered intradermally,subcutaneously, orally, intravenously, intravitreally (or otherwiseinjected into the eye), intra-arterially, intra-abdominally,intraperitoneally, intrathecally, or intramuscularly. In someembodiments, the pharmaceutical composition is administered using adevice implanted into the eye or other body part. In some embodiments,the subject is a mouse, rat, monkey, or human.

The pharmaceutical compositions can be administered for therapy by anysuitable route. In addition, the pharmaceutical compositions can alsocontain other pharmaceutically active compounds or a plurality ofcompounds of the invention.

In some embodiments, the pharmaceutical composition can be administeredsubcutaneously, orally, intramuscularly, or intravenously. In oneembodiment, the pharmaceutical composition is administered at atherapeutically effective dose. In some cases of the foregoing, thetherapeutically effective dose results in a gain in time spent within atherapeutic window for the fusion protein compared to the correspondingBP of the fusion protein not linked to the XTEN and administered at acomparable dose to a subject. The gain in time spent within thetherapeutic window can at least threefold greater than the correspondingBP not linked to the XTEN, or alternatively, at least four-fold, orfive-fold, or six-fold, or seven-fold, or eight-fold, or nine-fold, orat least 10-fold, or at least 20-fold greater than the corresponding BPnot linked to the XTEN.

In some embodiments, invention provides a method of treating a disease,disorder or condition, comprising administering the pharmaceuticalcomposition described above to a subject using multiple consecutivedoses of the pharmaceutical composition administered using atherapeutically effective dose regimen. In one embodiment of theforegoing, the therapeutically effective dose regimen can result in again in time of at least three-fold, or alternatively, at leastfour-fold, or five-fold, or six-fold, or sevenfold, or eight-fold, ornine-fold, or at least 10-fold, or at least 20-fold between at least twoconsecutive C_(max) peaks and/or C_(mm) troughs for blood levels of thefusion protein compared to the corresponding BP of the fusion proteinnot linked to XTEN(s) and administered at a comparable dose regimen to asubject. In some embodiments of the foregoing, the administration of thefusion protein results in a comparable improvement in at least onemeasured parameter using less frequent dosing or a lower total dosage inmoles of the fusion protein of the pharmaceutical composition comparedto the corresponding biologically active protein component(s) not linkedto XTEN(s) and administered to a subject using a therapeuticallyeffective regimen to a subject.

In one embodiment, the pharmaceutical composition is administeredsubcutaneously. In this embodiment, the composition may be supplied as alyophilized powder to be reconstituted prior to administration. Thecomposition may also be supplied in a liquid form or frozen, which canbe administered directly to a patient. In one embodiment, thecomposition is supplied as a liquid in a pre-filled syringe such that apatient can easily self-administer the composition.

Extended release formulations useful in the present invention may beoral formulations comprising a matrix and a coating composition.Suitable matrix materials may include waxes (e.g., camauba, bees wax,paraffin wax, ceresine, shellac wax, fatty acids, and fatty alcohols),oils, hardened oils or fats (e.g., hardened rapeseed oil, castor oil,beef tallow, palm oil, and soya bean oil), and polymers (e.g.,hydroxypropyl cellulose, polyvinylpyrrolidone, hydroxypropyl methylcellulose, and polyethylene glycol). Other suitable matrix tabletingmaterials are microcrystalline cellulose, powdered cellulose,hydroxypropyl cellulose, ethyl cellulose, with other carriers, andfillers. Tablets may also contain granulates, coated powders, orpellets. Tablets may also be multi-layered. Multi-layered tablets areespecially preferred when the active ingredients have markedly differentpharmacokinetic profiles. Optionally, the finished tablet may be coatedor uncoated.

The coating composition may comprise an insoluble matrix polymer and/ora water-soluble material. Water soluble materials can be polymers suchas polyethylene glycol, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinylpyrrolidone, polyvinyl alcohol, or monomericmaterials such as sugars (e.g., lactose, sucrose, fructose, mannitol andthe like), salts (e.g., sodium chloride, potassium chloride and thelike), organic acids (e.g., fumaric acid, succinic acid, lactic acid,and tartaric acid), and mixtures thereof. Optionally, an enteric polymermay be incorporated into the coating composition. Suitable entericpolymers include hydroxypropyl methyl cellulose, acetate succinate,hydroxypropyl methyl cellulose, phthalate, polyvinyl acetate phthalate,cellulose acetate phthalate, cellulose acetate trimellitate, shellac,zein, and polymethacrylates containing carboxyl groups. The coatingcomposition may be plasticised by adding suitable plasticisers such as,for example, diethyl phthalate, citrate esters, polyethylene glycol,glycerol, acetylated glycerides, acetylated citrate esters,dibutylsebacate, and castor oil. The coating composition may alsoinclude a filler, which can be an insoluble material such as silicondioxide, titanium dioxide, talc, kaolin, alumina, starch, powderedcellulose, MCC, or polacrilin potassium. The coating composition may beapplied as a solution or latex in organic solvents or aqueous solventsor mixtures thereof. Solvents such as water, lower alcohol, lowerchlorinated hydrocarbons, ketones, or mixtures thereof may be used.

BPXTEN polypeptides of the present invention can be formulated accordingto known methods to prepare pharmaceutically useful compositions,whereby the polypeptide is combined in admixture with a pharmaceuticallyacceptable carrier vehicle, such as aqueous solutions or buffers,pharmaceutically acceptable suspensions and emulsions. Examples ofnon-aqueous solvents include propyl ethylene glycol, polyethylene glycoland vegetable oils. Therapeutic formulations are prepared for storage bymixing the active ingredient having the desired degree of purity withoptional physiologically acceptable carriers, excipients or stabilizers,as described in Remington's Pharmaceutical Sciences 16th edition, Osol,A. Ed. (1980), in the form of lyophilized formulations or aqueoussolutions. The compositions of the invention may be formulated using avariety of excipients. Suitable excipients include microcrystallinecellulose (e.g. Avicel PH 102, Avicel PHlOl), polymethacrylate,poly(ethyl acrylate, methyl methacrylate, trimethylammonioethylmethacrylate chloride) (such as Eudragit RS-30D), hydroxypropylmethylcellulose (Methocel KlOOM, Premium CR Methocel KlOOM, Methocel E5,Opadry®), magnesium stearate, talc, triethyl citrate, aqueousethylcellulose dispersion (Surelease®), and protamine sulfate. The slowrelease agent may also comprise a carrier, which can comprise, forexample, solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents.Pharmaceutically acceptable salts can also be used in these slow releaseagents, for example, mineral salts such as hydrochlorides,hydrobromides, phosphates, or sulfates, as well as the salts of organicacids such as acetates, proprionates, malonates, or benzoates. Thecomposition may also contain liquids, such as water, saline, glycerol,and ethanol, as well as substances such as wetting agents, emulsifyingagents, or pH buffering agents. Liposomes may also be used as a carrier.

In some embodiments, the compositions of the present invention areencapsulated in liposomes, which have demonstrated utility in deliveringbeneficial active agents in a controlled manner over prolonged periodsof time. Liposomes are closed bilayer membranes containing an entrappedaqueous volume. Liposomes may also be unilamellar vesicles possessing asingle membrane bilayer or multilamellar vesicles with multiple membranebilayers, each separated from the next by an aqueous layer. Thestructure of the resulting membrane bilayer is such that the hydrophobic(non-polar) tails of the lipid are oriented toward the center of thebilayer while the hydrophilic (polar) heads orient towards the aqueousphase. In one embodiment, the liposome may be coated with a flexiblewater-soluble polymer that avoids uptake by the organs of themononuclear phagocyte system, primarily the liver and spleen. Suitablehydrophilic polymers for surrounding the liposomes include, withoutlimitation, PEG, polyvinylpyrrolidone, polyvinylmethylether,polymethyloxazoline, polyethyloxazoline, polyhydroxypropyloxazoline,polyhydroxypropylmethacrylamide, polymethacrylamide,polydimethylacrylamide, polyhydroxypropylmethacrylate,polyhydroxethylacrylate, hydroxymethylcellulose hydroxyethylcellulose,polyethyleneglycol, polyaspartamide and hydrophilic peptide sequences asdescribed in U.S. Pat. Nos. 6,316,024; 6,126,966; 6,056,973; 6,043,094,the contents of which are incorporated by reference in their entirety.

Liposomes may be comprised of any lipid or lipid combination known inthe art. For example, the vesicle-forming lipids may benaturally-occurring or synthetic lipids, including phospholipids, suchas phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid,phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, andsphingomyelin as disclosed in U.S. Pat. Nos. 6,056,973 and 5,874,104.The vesicle-forming lipids may also be glycolipids, cerebrosides, orcationic lipids, such as 1,2-dioleyloxy-3-(trimethylamino) propane(DOTAP);N-[1-(2,3,-ditetradecyloxy)propyl]-N,N-dimethyl-N-hydroxyethylammoniumbromide (DMRIE); N-[I-(2,3,-dioleyloxy)propyl]-N,N-dimethyl-N-hydroxyethylammonium bromide (DORIE);N-[I-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA); 3[N—(N′,N′-dimethylaminoethane) carbamoyl] cholesterol (DC-Choi); ordimethyldioctadecylammonium (DDAB) also as disclosed in U.S. Pat. No.6,056,973. Cholesterol may also be present in the proper range to impartstability to the vesicle as disclosed in U.S. Pat. Nos. 5,916,588 and5,874,104.

Additional liposomal technologies are described in U.S. Pat. Nos.6,759,057; 6,406,713; 6,352,716; 6,316,024; 6,294,191; 6,126,966;6,056,973; 6,043,094; 5,965,156; 5,916,588; 5,874,104; 5,215,680; and4,684,479, the contents of which are incorporated herein by reference.These describe liposomes and lipid-coated microbubbles, and methods fortheir manufacture. Thus, one skilled in the art, considering both thedisclosure of this invention and the disclosures of these other patentscould produce a liposome for the extended release of the polypeptides ofthe present invention. For liquid formulations, a desired property isthat the formulation be supplied in a form that can pass through a 25-,28-, 30-, 31-, 32-gauge needle for intravenous, intramuscular,intraarticular, or subcutaneous administration.

Administration via transdermal formulations can be performed usingmethods also known in the art, including those described generally in,e.g., U.S. Pat. Nos. 5,186,938 and 6,183,770, 4,861,800, 6,743,211,6,945,952, 4,284,444, and WO 89/09051, incorporated herein by referencein their entireties. A transdermal patch is a particularly usefulembodiment with polypeptides having absorption problems. Patches can bemade to control the release of skin-permeable active ingredients over a12 hour, 24 hour, 3 day, and 7 day period. In one example, a 2-folddaily excess of a polypeptide of the present invention is placed in anon-volatile fluid. The compositions of the invention are provided inthe form of a viscous, non-volatile liquid. The penetration through skinof specific formulations may be measures by standard methods in the art(for example, Franz et al., J. Invest. Derm. 64: 194-195 (1975)).Examples of suitable patches are passive transfer skin patches,iontophoretic skin patches, or patches with microneedles such asNicoderm. In other embodiments, the composition may be delivered viaintranasal, buccal, or sublingual routes to the brain to enable transferof the active agents through the olfactory passages into the CNS andreducing the systemic administration. Devices commonly used for thisroute of administration are included in U.S. Pat. No. 6,715,485.Compositions delivered via this route may enable increased CNS dosing orreduced total body burden reducing systemic toxicity risks associatedwith certain drugs. Preparation of a pharmaceutical composition fordelivery in a subdermally implantable device can be performed usingmethods known in the art, such as those described in, e.g., U.S. Pat.Nos. 3,992,518; 5,660,848; and 5,756,115.

Osmotic pumps may be used as slow release agents in the form of tablets,pills, capsules or implantable devices. Osmotic pumps are well known inthe art and readily available to one of ordinary skill in the art fromcompanies experienced in providing osmotic pumps for extended releasedrug delivery. Examples are ALZA's DUROS™; ALZA's OROS™; OsmoticaPharmaceutical's Osmodex™ system; Shire Laboratories' EnSoTrol™ system;and Alzet™. Patents that describe osmotic pump technology are U.S. Pat.Nos. 6,890,918; 6,838,093; 6,814,979; 6,713,086; 6,534,090; 6,514,532;6,361,796; 6,352,721; 6,294,201; 6,284,276; 6,110,498; 5,573,776;4,200,0984; and 4,088,864, the contents of which are incorporated hereinby reference. One skilled in the art, considering both the disclosure ofthis invention and the disclosures of these other patents could producean osmotic pump for the extended release of the polypeptides of thepresent invention.

Syringe pumps may also be used as slow release agents. Such devices aredescribed in U.S. Pat. Nos. 4,976,696; 4,933,185; 5,017,378; 6,309,370;6,254,573; 4,435,173; 4,398,908; 6,572,585; 5,298,022; 5,176,502;5,492,534; 5,318,540; and 4,988,337, the contents of which areincorporated herein by reference. One skilled in the art, consideringboth the disclosure of this invention and the disclosures of these otherpatents could produce a syringe pump for the extended release of thecompositions of the present invention.

Pharmaceutical Kits

In some embodiments, the invention provides a kit to facilitate the useof the BPXTEN polypeptides. In one embodiment, the kit comprises, in atleast a first container: (a) an amount of a BPXTEN fusion proteincomposition sufficient to treat a disease, condition or disorder uponadministration to a subject in need thereof, and (b) an amount of apharmaceutically acceptable carrier; together in a formulation ready forinjection or for reconstitution with sterile water, buffer, or dextrose;together with a label identifying the BPXTEN drug and storage andhandling conditions, and a sheet of the approved indications for thedrug, instructions for the reconstitution and/or administration of theBPXTEN drug for the use for the prevention and/or treatment of anapproved indication, appropriate dosage and safety information, andinformation identifying the lot and expiration of the drug. In someembodiments of the foregoing, the kit can comprise a second containerthat can carry a suitable diluent for the BPXTEN composition, which willprovide the user with the appropriate concentration of BPXTEN to bedelivered to the subject.

Method of Treatment

Disclosed herein includes use of a polypeptide, such as any describedhereinabove or described anywhere else herein, in the preparation of amedicament for the treatment of a disease in a subject. In someembodiments, the particular disease to be treated will depend on thechoice of the biologically active proteins. In some embodiments, thedisease is cancer (including any form thereof) In some cases, the canceror tumor can be characterized by a low, medium, or high level of HER2expression.

Disclosed herein includes a method of treating a disease in a subject,the method comprising administering to the subject in need thereof oneor more therapeutically effective doses of the pharmaceuticalcomposition, such as any described hereinabove or described anywhereelse herein. In some embodiments, the disease is cancer (including anyform thereof) In some embodiments, the subject is a mouse, rat, monkey,and human. In some cases, the cancer or tumor can be characterized by alow, medium, or high level of HER2 expression.

In certain embodiments, the HER-2 targeted bispecific compositions ofthe present invention (and particularly AMX818) may be advantageouslycombined with a second therapeutic agent effective for treating orameliorating the effects of the cancer. The additional therapeutic agentmay be selected from the group consisting of an antibody, an antibodyfragment, an antibody conjugate, a cytotoxic agent, a toxin, aradionuclide, an immunomodulator, a photoactive therapeutic agent, aradiosensitizing agent, a hormone, an anti-angiogenesis agent, andcombinations thereof. Particularly preferred second or additionaltherapeutic agents include other HER2 targeting agents, chemotherapyagents, radiotherapeutic agents, as well as agents that target HER3 andother targets that are involved in resistance to treatment ofHER2-driven cancers.

Examples of therapeutic antibodies that may be used in the presentinvention include rituximab (Rituxan), Brentuximab Vedotin (Adcetriz),Ado-trastuzumab emtansine (Kadcyla), Cetuximab (Erbitux), bevacizumab(Avastin), Ibritumomab (Zevalin), vedolizumab (Entyvio), Ipilimumab(Yervoy), Nivolumab (Opdivo), pembrolizumab (Keytruda), Alemtuzamabatezolizumab (Tecentriq), avelumab (Bavencio), durvalumab (Imfinzi),B-701, Ofatumumab, Obinutuzumab (Gazyva) Panitumumab, plozalizumab,BI-754091, OREG-103, COM-701, BI-754111, and combinations thereof.

According to some embodiments, the antibody, fragment thereof, orconjugate thereof is selected from the group consisting of rituximab(Rituxan), Brentuximab Vedotin (Adcetriz), Ado-trastuzumab emtansine(Kadcyla), Ipilimumab (Yervoy), Nivolumab (Opdivo), pembrolizumab(Keytruda), Alemtuzamab atezolizumab (Tecentriq), durvalumab (Imfinzi),Ofatumumab, Obinutuzumab (Gazyva) Panitumumab, and combinations thereof.

In other embodiments, the additional agent may be a DNA damaging agent,antimetabolite, anti-microtubule agent, antibiotic agent, etc. DNAdamaging agents include alkylating agents, platinum-based agents,intercalating agents, and inhibitors of DNA replication. Non-limitingexamples of DNA alkylating agents include cyclophosphamide,mechlorethamine, uramustine, melphalan, chlorambucil, ifosfamide,carmustine, lomustine, streptozocin, busulfan, temozolomide,pharmaceutically acceptable salts thereof, prodrugs, and combinationsthereof. Non-limiting examples of platinum-based agents includecisplatin, carboplatin, oxaliplatin, nedaplatin, satraplatin, triplatintetranitrate, pharmaceutically acceptable salts thereof, prodrugs, andcombinations thereof. Non-limiting examples of intercalating agentsinclude doxorubicin, daunorubicin, idarubicin, mitoxantrone,pharmaceutically acceptable salts thereof, prodrugs, and combinationsthereof. Non-limiting examples of inhibitors of DNA replication includeirinotecan, topotecan, amsacrine, etoposide, etoposide phosphate,teniposide, pharmaceutically acceptable salts thereof, prodrugs, andcombinations thereof. Antimetabolites include folate antagonists such asmethotrexate and premetrexed, purine antagonists such as6-mercaptopurine, dacarbazine, and fludarabine, and pyrimidineantagonists such as 5-fluorouracil, arabinosylcytosine, capecitabine,gemcitabine, decitabine, pharmaceutically acceptable salts thereof,prodrugs, and combinations thereof. Anti-microtubule agents includewithout limitation vinca alkaloids, paclitaxel (Taxol®), docetaxel(Taxotere®), and ixabepilone (Ixempra®). Antibiotic agents includewithout limitation actinomycin, anthracyclines, valrubicin, epirubicin,bleomycin, plicamycin, mitomycin, pharmaceutically acceptable saltsthereof, prodrugs, and combinations thereof.

Exemplary cytotoxic agents are know to those of skill in the art, andmay, for example, be selected from the group consisting ofcyclophosphamide, mechlorethamine, uramustine, melphalan, chlorambucil,ifosfamide, carmustine, lomustine, streptozocin, busulfan, temozolomide,cisplatin, carboplatin, oxaliplatin, nedaplatin, satraplatin, triplatintetranitrate, doxorubicin, daunorubicin, idarubicin, mitoxantrone,methotrexate, pemetrexed, 6-mercaptopurine, dacarbazine, fludarabine,5-fluorouracil, arabinosylcytosine, capecitabine, gemcitabine,decitabine, vinca alkaloids, paclitaxel (Taxol), docetaxel (Taxotere),ixabepilone (Ixempra), actinomycin, anthracyclines, valrubicin,epirubicin, bleomycin, plicamycin, mitomycin, pharmaceuticallyacceptable salts thereof, prodrugs, and combinations thereof.

Cytotoxic agents according to the present invention also include aninhibitor of the PI3K/Akt pathway. Non-limiting examples of an inhibitorof the PI3K/Akt pathway include A-674563 (CAS #552325-73-2), AGL 2263,AMG-319 (Amgen, Thousand Oaks, Calif), AS-041164(5-benzo[1,3]dioxol-5-ylmethylene-thiazolidine-2,4-dione), AS-604850(5-(2,2-Difluoro-benzo[1,3]dioxol-5-ylmethylene)-thiazolidine-2,4-dione),AS-605240 (5-quinoxilin-6-methylene-1,3-thiazolidine-2,4-dione), AT7867(CAS #857531-00-1), benzimidazole series, Genentech (Roche HoldingsInc., South San Francisco, Calif.), BML-257 (CAS #32387-96-5), BVD-723,CAL-120 (Gilead Sciences, Foster City, Calif), CAL-129 (GileadSciences), CAL-130 (Gilead Sciences), CAL-253 (Gilead Sciences), CAL-263(Gilead Sciences), CAS #612847-09-3, CAS #681281-88-9, CAS #75747-14-7,CAS #925681-41-0, CAS #98510-80-6, CCT128930 (CAS #885499-61-6),CH5132799 (CAS #1007207-67-1), CHR-4432 (Chroma Therapeutics, Ltd.,Abingdon, UK), FPA 124 (CAS #902779-59-3), GS-1101 (CAL-101) (GileadSciences), GSK 690693 (CAS #937174-76-0), H-89 (CAS #127243-85-0),Honokiol, IC87114 (Gilead Science), IPI-145 (Intellikine Inc.), KAR-4139(Karus Therapeutics, Chilworth, UK), KAR-4141 (Karus Therapeutics),KIN-1 (Karus Therapeutics), KT 5720 (CAS #108068-98-0), Miltefosine,MK-2206 dihydrochloride (CAS #1032350-13-2), ML-9 (CAS #105637-50-1),Naltrindole Hydrochloride, OXY-111A (NormOxys Inc., Brighton, Mass.),perifosine, PHT-427 (CAS #1191951-57-1), PI3 kinase delta inhibitor,Merck KGaA (Merck & Co., Whitehouse Station, N.J.), PI3 kinase deltainhibitors, Genentech (Roche Holdings Inc.), PI3 kinase deltainhibitors, Incozen (Incozen Therapeutics, Pvt. Ltd., Hydrabad, India),PI3 kinase delta inhibitors-2, Incozen (Incozen Therapeutics), PI3kinase inhibitor, Roche-4 (Roche Holdings Inc.), PI3 kinase inhibitors,Roche (Roche Holdings Inc.), PI3 kinase inhibitors, Roche-5 (RocheHoldings Inc.), PI3-alpha/delta inhibitors, Pathway Therapeutics(Pathway Therapeutics Ltd., South San Francisco, Calif.), PI3-deltainhibitors, Cellzome (Cellzome AG, Heidelberg, Germany), PI3-deltainhibitors, Intellikine (Intellikine Inc., La Jolla, Calif), PI3-deltainhibitors, Pathway Therapeutics-1 (Pathway Therapeutics Ltd.),PI3-delta inhibitors, Pathway Therapeutics-2 (Pathway TherapeuticsLtd.), PI3-delta/gamma inhibitors, Cellzome (Cellzome AG),PI3-delta/gamma inhibitors, Cellzome (Cellzome AG), PI3-delta/gammainhibitors, Intellikine (Intellikine Inc.), PI3-delta/gamma inhibitors,Intellikine (Intellikine Inc.), PI3-delta/gamma inhibitors, PathwayTherapeutics (Pathway Therapeutics Ltd.), PI3-delta/gamma inhibitors,Pathway Therapeutics (Pathway Therapeutics Ltd.), PI3-gamma inhibitorEvotec (Evotec), PI3-gamma inhibitor, Cellzome (Cellzome AG), PI3-gammainhibitors, Pathway Therapeutics (Pathway Therapeutics Ltd.), PI3Kdelta/gamma inhibitors, Intellikine-1 (Intellikine Inc.), PI3Kdelta/gamma inhibitors, Intellikine-1 (Intellikine Inc.), pictilisib(Roche Holdings Inc.), PIK-90 (CAS #677338-12-4), SC-103980 (Pfizer, NewYork, N.Y.), SF-1126 (Semafore Pharmaceuticals, Indianapolis, Ind.),SH-5, SH-6, Tetrahydro Curcumin, TG100-115 (Targegen Inc., San Diego,Calif), Triciribine, X-339 (Xcovery, West Palm Beach, Fla.), XL-499(Evotech, Hamburg, Germany), pharmaceutically acceptable salts thereof,and combinations thereof.

The additional agent in the combination therapy may be a poison or venomof plant or animal origin. An example is diphtheria toxin or portionsthereof. In other examples, the additional agent may be a “radionuclide”i.e., a radioactive substance administered to the patient, e.g.,intravenously or orally, after which it penetrates via the patient'snormal metabolism into the target organ or tissue, where it deliverslocal radiation for a short time. Examples of radionuclides include, butare not limited to, I-125, At-211, Lu-177, Cu-67, I-131, Sm-153, Re-186,P-32, Re-188, In-114m, and Y-90.

The term “immunomodulator” means a substance that alters the immuneresponse by augmenting or reducing the ability of the immune system toproduce antibodies or sensitized cells that recognize and react with theantigen that initiated their production. Immunomodulators may berecombinant, synthetic, or natural preparations and include cytokines,corticosteroids, cytotoxic agents, thymosin, and immunoglobulins. Someimmunomodulators are naturally present in the body, and certain of theseare available in pharmacologic preparations. Examples ofimmunomodulators include, but are not limited to, granulocytecolony-stimulating factor (G-CSF), LAG-3, IMP-321, JCAR-014, ASLAN-002(BMS-777607), interferons, imiquimod and cellular membrane fractionsfrom bacteria, IL-2, IL-7, IL-12, CCL3, CCL26, CXCL7, synthetic cytosinephosphate-guanosine (CpG), immune-checkpoint inhibitors, andcombinations thereof. Targeted cytokine therapy, particularly targetingto lymphotoxin and LiGHT may also be useful in combination with thecompositions of the present invention.

In some combination treatments, the additional agent may be a“radiosensitizing agent” that makes tumor cells more sensitive toradiation therapy. Examples of radiosensitizing agents includemisonidazole, metronidazole, tirapazamine, and trans sodium crocetinate,and combination thereof.

In still other embodiments, the additional agent is an“anti-angiogenesis” agent that reduces or inhibits the growth of newblood vessels, such as, e.g., an inhibitor of vascular endothelialgrowth factor (VEGF) and an inhibitor of endothelial cell migration.Anti-angiogenesis agents include without limitation 2-methoxyestradiol,angiostatin, bevacizumab, cartilage-derived angiogenesis inhibitoryfactor, endostatin, IFN-α, IL-12, itraconazole, linomide, plateletfactor-4, prolactin, SU5416, suramin, tasquinimod, tecogalan,tetrathiomolybdate, thalidomide, thrombospondin, thrombospondin,TNP-470, ziv-aflibercept, pharmaceutically acceptable salts thereof,prodrugs, and combinations thereof.

In particularly preferred embodiments, the HER2-targeted bispecificcompositions of the present invention may be combined with checkpointinhibitors, such as anti-PD-1/anti-PD-L1 compositions, CTLA-4, OX40 andthe like. In particular embodiments of such combination therapy, thecompositions of the present invention can be combined with antagonistsof the cell surface receptor programmed cell death protein 1, also knownas PD-1 as well as, antagonists of PD-L1.

PD-1 plays an important role in down-regulating the immune system andpromoting self-tolerance by suppressing T cell inflammatory activity.Binding of the PD-1 ligands, PD-L1 and PD-L2 to the PD-1 receptor foundin T cells inhibits T-cell proliferation and cytokine production.Upregulation of PD-1 ligands occurs in some tumors and signaling throughthis pathway can contribute to inhibition of active T-cell immunesurveillance of tumors. Anti-PD-1 antibodies bind to the PD-1 receptorand block its interaction with PD-L1 and PD-L3, releasing PD-1pathway-mediated inhibition of the immune response, including theanti-tumor immune response.

Those of skill in the art are aware of various anti-PD-1 antibodies thatmay be used. In some embodiments, an exemplary anti-PD-1 antibody usedin combination with the compounds of the present invention isPembrolizumab (Keytruda®). In other embodiments, the anti-PD-1 antibodyused in combination with the compound described above is Nivolumab(Opdivo®). In other embodiments, the anti-PD-1 antibody used incombination with the compound described above is Pidilizumab(Medivation).

Additional PD-1 antibodies known to those of skill in the art, includeAGEN-2034 (Agenus), AMP-224 (Medimmune), BCD-100 (Biocad), BGBA-317(Beigene), BI-754091 (Boehringer Ingelheim), CBT-501 (Genor Biopharma),CC-90006 (Celgene), cemiplimab (Regeneron Pharmaceuticals),durvalumab+MEDI-0680 (Medimmune), GLS-010 (Harbin GloriaPharmaceuticals), IBI-308 (Eli Lilly), JNJ-3283 (Johnson & Johnson),JS-001 (Shanghai Junshi Bioscience Co.), MEDI-0680 (Medimmune), MGA-012(MacroGenics), MGD-013 (Marcogenics), pazopanibhydrochloride+pembrolizumab (Novartis), PDR-001 (Novartis), PF-06801591(Pfizer), REGN-2810 (Regeneron), SHR-1210 (Jiangsu Hengrui MedicineCo.), TSR-042 (Tesaro Inc.), LZM-009 (Livzon Pharmaceutical Group Inc)and ABBV-181 (AbbVie Inc). Each possibility represents a separateembodiment of the present invention.

In one preferred embodiment for combination therapy of the presentinvention, the anti-PD-1 antibody is pembrolizumab (Keytruda®).

In other embodiments, the compositions of the present invention arecombined with an anti-PD-L1 antibody. Exemplary such anti-PD-L1antibodies used in the combinations of the present invention may beselected from the group consisting of Durvalumab (MedImmune LLC),Atezolizumab (Hoffmann-La Roche Ltd, Chugai Pharmaceutical Co Ltd),Avelumab (Merck KGaA), CX-072 (CytomX Therapeutics Inc), BMS-936559(ViiV Healthcare Ltd), SHR-1316 (Jiangsu Hengrui Medicine Co Ltd),M-7824 (Merck KGaA), LY-3300054 (Eli Lilly and Co), FAZ-053 (NovartisAG), KN-035 (AlphaMab Co Ltd), CA-170 (Curis Inc), CK-301 (TGTherapeutics Inc), CS-1001 (CStone Pharmaceuticals Co Ltd), HLX-10(Shanghai Henlius Biotech Co Ltd), MCLA-145 (Merus NV), MSB-2311(MabSpace Biosciences (Suzhou) Co Ltd) and MEDI-4736 (Medimmune).

In certain other embodiments, the combination therapies of the presentinvention may include other HER2-direct therapies. For example, it maybe advantageous to combine the compositions of the present inventionwith trastuzumab and related HER2 based (i.e., those that target thetrastuzumab epitope) therapies such as Trastuzumab, Trastuzumab basedADCs, Kadcyla, Enhertu, HER2 tyrosine kinase inhibitors such aslapatinib, neratinib, tucatinib, HER2 targeting immunoconjugates, suchas TLRs, and cytokines. Other immunotherapies and checkpointinhibitor-based therapies that may be useful in combination with thecompositions of the present invention include CTLA4, TIGIT, OX40, PD1,PDL1, TIM3-based therapies. The compositions of the invention mayfurther be combined with CAR-T, NK, or T-cell based therapies, as wellas with immunotherapy vaccines. Combination with cytokines and therapiesthat are cytokine targeting also are contemplated. Of particularinterest may be use of TGFb, VEGF and VEGFR1-3 and anti-angiogenesistargeting therapies such as Avastin, Ramucirumab, or tyrosine kinaseinhibitors such as axitinib, lenvatinib, cabozantinib, regorafenib,sunitib, sorafenib. In other embodiments, CDK4/6 inhibitors such aspalbociclib and the like may be used. EGFR inhibitors such as cetuximab,panitumumab, erlotinib, and Osimertinib also may be of use incombination therapies.

The following are examples of compositions and evaluations ofcompositions of the disclosure. It is understood that various otherembodiments may be practiced, given the general description providedabove.

EXAMPLES Example 1. Design of Barcoded XTEN by Minimal Mutations fromGeneral-Purpose XTEN

This example illustrates an exemplary design approach to barcoded XTENby making minimal mutation(s) of the amino acid sequence of ageneral-purpose XTEN (such as one of Table 3b hereinabove). The relevantcriteria for performing minimal mutation(s) include one or more of thefollowing: (a) to minimize the sequence change of the correspondingXTEN; (b) to minimize the amino acid composition change in thecorresponding XTEN; (c) to substantially maintain the net charge in thecorresponding XTEN; (d) to substantially maintain the low immunogenicityof the corresponding XTEN; (e) to substantially maintain thepharmacokinetic properties afforded by the XTEN.

For example, barcoded XTENs were constructed by performing one or moremutations comprising deletion of a glutamic acid residue, insertion of aglutamic acid residue, substitution of a glutamic acid residue, orsubstitution for a glutamic acid residue, or any combination thereof tothe general-purpose XTENs in Table 9.

TABLE 9 Four general-purpose XTENs used for engineering of barcoded XTENSEQ ID XTEN NO. Name Amino Acid Sequence 676 AE144GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAP 677 AE288_1GTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEG SAP 678 AE576GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEP SEGSAP 679AE864 GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTS TEPSEGSAP

Example 2. Sequence Analysis of Barcoded XTEN and Selection Thereof forFusion to a Biologically-Active Polypeptide (“BP”)

This example illustrates the design and selection of barcoded XTEN (andthe assembly of more than one barcoded XTENs into a set) for fusion to abiologically-active polypeptide. Depending on the location of thebarcode fragment(s) within the XTEN(s) and the manner in which theXTEN(s) is/are fused to a biologically-active protein to form anXTEN-containing construct (e.g., an XTENylated protease-activated T-cellengager (XPAT)), the barcode fragment(s) can indicate truncation(s) ofthe XTEN.

In silico GluC digestion analysis was performed on two exemplary XTENs(XTEN864 and XTEN288_1) to identify the releasable peptide fragmentsupon complete GluC digestion of the XTEN. The in silico analysis willtake into consideration that, with respect to the XTEN havingconsecutive glutamic acid residues (e.g., “EE”), GluC may cleave aftereither one of the glutamic acid residues. As shown in the resultssummarized below in Table 10, a 10-mer peptide sequence “TPGTSTEPSE”(SEQ ID NO: 96) and a 14-mer peptide sequence “GSAPGSEPATSGSE” (SEQ IDNO: 97) each occur once and only once in the longer XTEN864, while allother peptide sequences occur two or more times in XTEN864. And the14-mer peptide sequence “GSAPGSEPATSGSE” (SEQ ID NO: 97) also occursonce and only once in the shorter XTEN288_1.

The uniqueness of a candidate barcode is assessed in relation to allother peptide fragments releasable from the XTEN-containing construct.Accordingly, a barcode sequence in one XTEN cannot occur anywhere elsein the XTEN-containing construct, including any other XTEN containedtherewithin, any biologically-active protein contained therewithin, orany connection between neighboring components thereof. For example,Table 11 shows a peptide “uniqueness” table for the set of two XTEN. Dueto its presence in both XTEN864 and XTEN288, the 14-mer peptide sequence“GSAPGSEPATSGSE” (SEQ ID NO: 97) is not unique to the set of XTENcomprising both XTEN864 and XTEN288 and, thus, may not be used as abarcode for detecting truncations in polypeptide products that containboth of the two XTEN sequences.

The selection of a barcode (or a set of barcodes) may further involveidentifying and determining the proper location(s) or position(s) of thecandidate barcode(s) within the XTEN. The location or position of acandidate barcode can be associated with pharmacologically relevantinformation of the XTEN (and the XTEN-containing construct as a whole),such as truncation of the XTEN beyond a critical length and/ordeletion(s) in the XTEN sequence. The 10mer peptide “TPGTSTEPSE” (SEQ IDNO: 96) could serve as a suitable barcode fragment if XTEN864 is placedat the N-terminus of the XTEN-containing product and if the truncationof 238 amino acids from the N-terminus of the product does notsignificantly impact the pharmacological properties of the product.

TABLE 10Representative XTEN sequences for in silico GluC digestion analysisExemplary XTEN Amino acid sequence XTEN864GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP (SEQ ID NO: 98) AE288_1GTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP (SEQ ID NO: 99)

TABLE 11 Peptide “uniqueness” analysis Peptide SEQ ID XTEN fragments NO:AE288 1 XTEN864 Both SATPE 125 9 23 32 SGPGSEPATSGSE 126 4 9 13GSAPGTSTEPSE 127 1 10 11 TPGTSE 128 4 7 11 SGPGTSTEPSE 129 2 8 10GSAPGTSE 130 1 8 9 GTSTEPSE 131 2 5 7 GSAPGSPAGSPTSTEE 132 1 4 5GSPAGSPTSTEE 133 1 4 5 SGPGTSE 134 2 3 5 SGPGSPAGSPTSTEE 135 1 3 4TPGSEPATSGSE 136 1 2 3 TPGSPAGSPTSTEE 137 1 2 3 GSAPGSEPATSGSE 138 1 1 2TPGTSTEPSE 139 1 1 All underlined sequences produce unique GluC peptidesNon-XTEN core underlined and italic Barcode peptides are bold

Exemplary barcode peptide sequences are illustrated below in Table 12.These barcode sequences should be flanked according to the structuralformula (I):

  AAA-Glu-Barcode Peptide-BBB,wherein “AAA” represents Gly, Ala, Ser, Thr or Pro and “BBB” representGly, Ala, Ser, or Thr configured to facilitate efficient release of thebarcode peptide by GluC digestion. Notably, the insertion of eachbarcode peptide in the XTEN may result in additional unique sequencesdirectly preceding or following the inserted barcode peptides.

TABLE 12 List of non-limiting examples of barcode peptides Candidate SEQBarcode ID Peptide(s) NO: SPATSGSTPE 140 GSAPATSE 141 GSAPGTATE 142GSAPGTE 143 PATSGPTE 144 SASPE 145 PATSGSTE 146 GSAPGTSAE 147 SATSGSE148 SGPGSTPAE 149

Example 3: Design and Selection of XTEN(s) in Full Sequence XTENylatedPolypeptide Constructs

This example illustrates the design of a full-sequence polypeptideconstruct, containing two XTENs, one at the N-terminus and the other atthe C-terminus.

Table 13 below illustrates XTEN sequences used in a representativebarcoded BPXTEN (containing barcoded XTENs at both the N- and C-termini)and a reference BPXTEN (containing general-purpose XTENs at both the N-and C-termini). In the representative barcoded BPXTEN, a barcoded XTEN(SEQ ID No. 8014) is fused at the N-terminus of the BP, and anotherbarcoded XTEN (SEQ ID No. 8015) is fused at the C-terminus of the BP. Inthe reference BPXTEN, a “Ref-N” XTEN is fused at the N-terminus of theBP, and a “Ref-C” XTEN is fused at the C-terminus of the BP. The “Ref-N”XTEN is comparable in length to the barcoded XTEN SEQ ID No. 8014; andthe “Ref-C” XTEN is comparable in length to the barcoded XTEN SEQ ID No.8015. The barcoded and reference BPXTENs each contain a referencesequence in the BP component. The reference sequence is unique anddiffers in molecular weight from all other peptide fragments that arereleasable from the corresponding BPXTEN upon complete digestion by GluCprotease (e.g., according to Example 5). The uniqueness of the referencesequence is assessed in relation to all other peptide fragmentsreleasable from the BPXTEN construct.

TABLE 13 Representative sets of N- and C-terminal XTENs usedin full-length BPXTEN constructs SEQ ID XTEN Total # NO. TypeAmino Acid Sequence of AAs 8014 N-terminalSPAGSPTSTESGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPAT 292 (from XTENSGSETPGTSESATPE SGPGSTPAESGSE TPGTSESATPESGPGTSTEPSEGSA Table 3a)PGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGGSAP 8015 C-terminalPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTST 582 (from XTENEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEG Table 3a)SAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSE GSAPGTESTPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGEPEA 8021 N-terminalSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPAT 292 Ref-N XTENSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGGSAP 8022 C-terminalPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTST 584 Ref-C XTENEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGAAEPE A *Barcodepeptides are bold All underlined sequences produce unique GluC peptides;

Example 4: Recombinant Construction and Production of BarcodedXTENylated Fusion Polypeptides

Example 4 illustrates recombinant construction, production, andpurification of an XTENylated fusion polypeptide containing a barcodedXTEN at the C-terminus and another barcoded XTEN at the N-terminus usingthe methods disclosed herein.

EXPRESSION: A construct encoding an XTENylated fusion polypeptide thatcontains an anti-CD3 sequence (e.g., set forth in Tables 6a-6e), ananti-HER2 sequence (e.g., set forth in Table 6f), a barcoded XTEN (e.g.,set forth in Table 3a) at the C-terminus, and a barcoded XTEN (e.g., setforth in Table 3a) at the N-terminus is expressed in a proprietary E.coli AmE098 strain and partitioned into the periplasm via an N-terminalsecretory leader sequence (MKKNIAFLLASMFVFSIATNAYA) (SEQ ID NO: 100),which is cleaved during translocation. Fermentation cultures are grownwith animal-free complex medium at 37° C. and temperature shifted to 26°C. prior to phosphate depletion. During harvest, fermentation wholebroth is centrifuged to pellet the cells. At harvest, the total volumeand the wet cell weight (WCW; ratio of pellet to supernatant) arerecorded, and the pelleted cells are collected and frozen at −80° C.

RECOVERY: The frozen cell pellet is resuspended in Lysis Buffer (100 mMcitric acid) targeting 30% wet cell weight. The resuspension is allowedto equilibrate at pH 4.4 then homogenized at 17,000±200 bar while outputtemperature is monitored and maintained at 15±5° C. The pH of thehomogenate is confirmed to be within the specified range (pH 4.4±0.1).

CLARIFICATION: To reduce endotoxin and host cell impurities, thehomogenate is allowed to undergo low-temperature (10±5° C.), acidic (pH4.4±0.1) flocculation overnight (15-20 hours). To remove the insolublefraction, the flocculated homogenate is centrifuged for 40 minutes at8,000 RCF and 2-8° C., and the supernatant is retained. To removenucleic acid, lipids, and endotoxin and to act as a filter aid, thesupernatant is adjusted to 0.1% (m/m) diatomaceous earth. To keep thefilter aid suspended, the supernatant is mixed via impeller and allowedto equilibrate for 30 minutes. A filter train, consisting of a depthfilter followed by a 0.22 μm filter, is assembled then flushed with MQ.The supernatant is pumped through the filter train while modulating flowto maintain a pressure drop of 25±5 psig.

Purification

Protein-L Capture: To remove host cell proteins, endotoxin, and nucleicacid, Protein-L is used to capture the kappa domain present within theaHER2 scFv of the BPXTEN molecule. The Protein-L stationary phase (TosohTP AF-rProtein L-650F), Protein-L mobile phase A (11.5 mM citric acid,24.5 mM Na₂HPO₄, 125 mM NaCl, 0.005% polysorbate 80, pH 5.0), andProtein-L mobile phase B (11 mM phosphoric acid, 0.005% polysorbate 80,pH 2.0) are used herein. The column is equilibrated with Protein-Lmobile phase A. The filtrate is adjusted to pH 5.5±0.2 and loaded ontothe Protein-L column targeting 2-4 g/L-resin then chased with Protein-Lmobile phase A until absorbance at 280 nm (A280) returns to (local)baseline. Bound material is eluted with mobile phase B and collected asa 2 CV fraction pre-spiked with 0.4 CV of 0.5 M Na₂HPO₄ and is analyzedby SDS-PAGE.

C-tag Intermediate Purification: To ensure C-terminal integrity, C-tagAffinity Chromatography is used to capture the C-terminal-EPEA tag. TheC-tag stationary phase (Thermo C-tagXL), C-tag mobile phase A (50 mMhistidine, 200 mM NaCl, 0.005% polysorbate 80, pH 6.5), and C-tag mobilephase B (20 mM Tris, 0.6 M MgCl₂, 0.005% polysorbate 80, pH 7.0) areused herein. The column is equilibrated with C-tag mobile phase A. TheIMAC Elution is loaded onto the C-tag column targeting 2 g/L-resin andchased with C-tag mobile phase A until absorbance at 280 nm (A280)returned to (local) baseline. Bound material is eluted with a C-tagmobile phase B. The C-tag Elution is collected as a 2 CV fraction and isanalyzed by SDS-PAGE.

AEX Polishing: To separate dimer and aggregate from monomeric productAnion Exchange (AEX) chromatography is utilized to capture theelectronegative N- and C-terminal XTEN domains. The AEX1 stationaryphase (BIA QA-80), AEX1 mobile phase A (50 mM histidine, 200 mM NaCl,0.005% polysorbate 80, pH 6.5), and AEX1 mobile phase B (50 mMhistidine, 500 mM NaCl, 0.005% polysorbate 80, pH 6.5) are used herein.The column is equilibrated with AEX mobile phase A. The C-tag elution isdiluted to 10 mS/cm with MQ, loaded targeting 2 g/L-resin, and thenchased with AEX mobile phase A until absorbance at 280 nm returned to(local) baseline. Bound material is eluted with a gradient from 0% B to100% B over 60 CV. Fractions are collected in 1 CV aliquots while A280≥2mAU above (local) baseline. Elution fractions are analyzed by SDS-PAGEand SE-HPLC, and fractions found to be ≥98% monomer are pooled (AEXPool) for further processing.

FORMULATION: To exchange the product into formulation buffer and tobring the product to the target concentration (0.5 g/L),Ultrafiltration/Diafiltration (UF/DF) is used. Using a 10 kDa membranewith an area of 0.1 m² and a TMP target of 15 psi, the AEX pool isconcentrated to 0.5 g/L, then diluted 10-fold with Formulation Buffer(50 mM histidine, 200 mM NaCl, 0.005% polysorbate 80, pH 6.5). The AEXpool is concentrated 10-fold and diluted 10-fold two more times. Therecovered Formulated product is 0.22 μm filtered within a BSC,aliquoted, labeled, and stored at −80° C. as Bulk Drug Substance (BDS).The BDS is confirmed by various analytical methods to meet all LotRelease criteria. Overall quality is analyzed by SDS-PAGE, the ratio ofmonomer to dimer and aggregate is analyzed by SE-HPLC, and N-terminalquality and product homogeneity is analyzed by HI-HPLC. Identity isconfirmed by ESI-MS.

Example 5. Release of Barcode Peptides by Protease Digest

This example illustrates the release of barcode fragment(s) andreference fragment(s) from a polypeptide mixture that contain varyinglengths or truncated forms of the XTEN-containing construct using themethods disclosed herein.

A sample of XTEN-containing construct is reduced and alkylated viaincubation in DTT and then iodoacetamide, sequentially. The samples arethen buffer exchanged and desalted using a size-exclusion spincartridge. Glu-C protease is added to the samples at an enzyme tosubstrate ratio of 1:5 and the samples are incubated at 37° C. fordigestion. Samples are then moved to 4° C. to halt the proteolyticreaction and placed in autosampler vials for analysis.

Example 6. Detection and Quantification of Barcode Peptide(s) andReference Peptide(s)

This example illustrates mass spectrometry methods used to generatequantitative measurements of individual barcode peptides. An LC-ParallelReaction Monitoring (PRM) method is programmed into a high-resolutionaccurate mass (HRAM) mass spectrometer. Unlike traditionalData-Dependent Acquisition (DDA) mass spectrometry methods, PRM methodsfocus on a specific set of 15-30 peptides in one run, sequencing each byMS-MS once per duty cycle. As such, this method generates eXtracted IonChromatograms (XICs) for the unfragmented precursor ions of the intactpeptide, as well as for each fragment ion of the peptide to confirm itssequence. Fragment ion XICs are often more sensitive and selectivelyquantitative than the precursor ion fragments. The LC-PRM method usedincludes the light and heavy versions of seven barcode peptides.Chromatographic peak areas of all fragment ions of these 14 peptides aremeasured post-acquisition and the strongest fragment ion is used forquantitative measurement. Peak area ratios of the XTEN barcode peptidesto the PAT barcode peptides are then calculated for relative XTEN:PATabundance at various points across the XTEN molecules.

Example 7. Stable Isotope Labeling to Quantify the Peptides by MassSpectrometry (MS)

This example illustrates the stable-isotope labeling schema to enableabsolute (rather than relative) quantitation of barcode peptides fromXTEN-containing polypeptides. A standard Heavy labeled Amino acidquantitative schema will be employed wherein synthetic analogues ofbarcode peptides in which the C-terminal Glutamic Acid is replaced withthe (¹³C)₅H₇(¹⁵N)O₃ heavy labeled analogue are procured from aspecialized vendor. A calibration curve will be prepared where a knownamount of XTEN barcode containing polypeptide is serially diluted into amatrix where the heavy-labeled synthetic peptide is held at a constantconcentration. Accurate quantitation can be performed by calibratingchromatographic peak area heavy:light ratios from the curve againstresearch samples containing the same spike-level of heavy labeledpeptide.

Example 8. Quantification of Truncation of XTEN-Containing Polypeptide

This example illustrates the quantification of length variants ortruncation variants in a mixture of XTEN-containing polypeptides.

For example, a barcode peptide “SGPGSTPAESGSE” (SEQ ID NO: 150), ispositioned 76 amino acids into the representative barcoded BPXTENsequence described in and obtained from Example 3 to indicate a severetruncation of the XTEN at the N-terminal end of the BPXTEN. Alsoconsider a potential barcode fragment “SPAGSPTSTESGTSE” (SEQ ID NO:151), is positioned at the N-terminus. The abundance measurement ratioof each barcode peptide relative to a unique reference peptide sequencefrom the biologically active protein (e.g., an scFv fragment) sequencefollowing the procedure of Example 6 indicates the total amount of thefull-length polypeptides and the variants having truncations that couldaffect pharmacological efficacy in the sample mixture. The abundancemeasurement of at least one reference fragment is used to indicate thetotal amount of all variants of the polypeptide in the sample mixture.Accordingly, differential abundance between the reference fragment andthe barcode fragments informs the amount of truncated polypeptidevariants. The LC-MS data are analyzed to determine the ratio of theamount of the barcode fragment to the reference fragment, indicating therelative amount of pharmacologically-efficacious variants in thepolypeptide mixture.

A set of two (or three) barcodes are used to indicate different levelsof truncation of the polypeptide. The LC-MS data are used to determinethe ratio of the amount of each barcode fragment to the amount of thereference fragment, thereby quantifying the distribution of truncationvariants in the polypeptide mixture.

Example 9. In Vitro Cytotoxicity of XTENylated (Masked) andDe-XTENylated (Unmasked, Activated) XPATs (Protease-Activated T CellEngager) Against Target Cells

This example illustrates masking by XTENs on XTENylatedProtease-Activated T-cell engagers (“XPATs”) in general, and onHER2-XPATs in particular. This example illustrates the differential incytotoxicity of XTENylated (masked) HER2-XPATs (e.g., as set forth inTable D) and the corresponding de-XTENylated (unmasked) HER2-PATs.

The cytotoxicity of an XTENylated PAT (as set forth in Table D) and thecorresponding de-XTENylated PAT (protease-treated) was determined usingan in vitro cytotoxicity assay which utilized the amount of ATP presentin wells of lysed target cells post treatment as a proxy for measuringcell viability. HER2-expressing target cells were seeded on white,opaque bottom plates at varying densities (BT474: 20k cells/well, andSKOV3: 10k cells/well) and allowed to incubate at 37° C., 5% CO₂overnight (18-24 hours). Prior to the end of the overnight incubation,peripheral blood mononuclear cells (PBMCs) were thawed and incubated at37° C., 5% CO₂ overnight. The PBMCs were isolated from screened, healthydonors by Ficoll density gradient centrifugation from either whole bloodor from lymphocyte-enriched buffy coat preparations obtained fromBioIVT. 10× XTENylated and de-XTENylated PAT titrations were preparedusing a 9-point, 3-fold titration (10th point is non-treatment) with astarting concentration of 2400 nM for the XTENylated PAT and 10 nM forthe de-XTENylated PAT. The PBMCs were seeded in the wells at 1:1Effector:Target ratio. 10× XTENylated and de-XTENylated PAT titrationswere diluted 10-fold into the wells for starting concentrations of 240nM and 1 nM, respectively. The plates were incubated at 37° C., 5% CO₂for 48 hours. After the 48-hour incubation, the plates were washed 3×with 1×PBS, and 100 μL of 1×PBS was added to all wells. 100 μL ofCellTiter-Glo® luminescent substrate solution was added to all wells,and the plates were allowed to incubate at room temperature for 1-5minute(s). The plates were then shaken on a plate shaker at 300-500 rpmfor 30-60 seconds to mix the contents of the wells and then read in aluminometer using an integration time of 100 ms. The intensity of signalproduced correlates to the amount of viable cells present in the wells.The mean of the signal from all non-treatment wells was calculated andused to determine % Live cells from treatment wells ((TreatmentSignal/Mean of Non-Treatment Signal)*100=% Live). The % Live was plottedby concentration, and half maximal response (EC50) values were derivedwith a 4-parameter logistic regression equation using GraphPad Prismsoftware.

The XTENylated PAT and the corresponding de-XTENylated PAT displayedlarge differences in cytotoxicity on all HER2-expressing cell linestested, confirming XTENylation results in reduction of cytolyticactivity of the masked bispecific antibody (inactivated state). As shownin FIGS. 5A-5B, cytotoxicity of de-XTENylated (unmasked, activated) PATon BT-474 and SK-OV-3 cells was observed in a dose-dependent manner,with maximal killing of ˜80% observed at 0.3 nM. As shown in FIGS.5A-5B, the de-XTENylated PAT exhibited EC50 values of 4.8 μM (BT474) and3.4 μM (SKOV3), while the XTENylated PAT exhibited EC50 values of 49,370μM (BT474) and 44,474 μM (SKOV3). The observations corroborate thatde-XTENylated HER2-PAT has cytotoxic activity on cell lines with HER2expression, and XTENylation masks (or shields) the ability to form animmune synapse, resulting in a reduction of cytotoxicity.

Example 10. In Vitro Cytotoxicity of XTENylated (Masked) andDe-XTENylated (Unmasked, Activated) XPATs Against Target Cells thatExpress Low Levels of Target Antigen Example 10a

Normal human cardiomyocytes express low levels of HER2 and as a result,rare cases of cardiac toxicity have been observed in patients treatedwith some HER2-targeted therapies. Given the known potentialcardiotoxicity of HER2-targeted agents, AMX-818 toxicity against humancardiomyocytes which express HER2 was assessed. Consistent with the lackof cardiotoxicity in non-human primate (NHP) preclinical studies,AMX-818 showed limited cytotoxicity against human cardiomyocytes invitro, even at micromolar doses. In contrast, the unmasked TCEcounterpart demonstrated significantly greater cytotoxicity activity(EC50˜100 pM)

This example illustrates the sensitivity of primary cardiomyocytes to Tcell-directed cytotoxicity in response to increasing concentrations of aHER2-XPAT (e.g., set forth in Table D) and its active proteolyticproduct, the corresponding HER2-PAT.

Normal human cardiomyocytes purchased from FujiFilm Cellular Dynamicswere used. Cardiomyocytes (iCell cardiomyocytes, Cellular DynamicsInternational) were revived from liquid nitrogen and plated at 20,000cells per 96-well for 7 days and treated as per the manufacturer'sinstructions. Human peripheral blood lymphocytes were added onto theiCell cardiomyocytes at a 1:1 Effector:Target ratio with increasing3-fold concentrations of the XTENylated HER2-XPAT or the correspondingde-XTENylated HER2-PAT and incubated for 48 hours at 37° C., 5% CO₂. Theassay was performed in RPMI (Roswell Park Memorial Institute) medium and10% heat-inactivated fetal bovine serum. Cardiomyocyte cell viabilitywas determined via ATP quantification and was performed with the CellTiter-Glo Luminescent Cell Viability Assay System (Promega). Cellsupernatant was aspirated, and cells were washed twice with phosphatebuffered saline (PBS), aspirated and followed by addition of PBS (100 μlper well). Automated plate washing was carried out using an LS405microplate washer dispenser (BioTek). Cell Titer-Glo reagent was added(100 μl per well), and assay plates were incubated for 5 minutes at roomtemperature. Luminescence was quantified with a multi label reader(Molecular Devices) with a luminescence detector. For analysis ofcytotoxicity, % viable cells was calculated from relative luminescenceunits (RLU). % live=(Test well RLU/Target cell only RLU)*100. For EC₅₀determination, data were transformed in Microsoft Excel and analyzedwith Graph Pad Prism 8.3.1 software ‘log(agonist) vs. response-variableslope (four parameters).

As shown in FIG. 6A, the cardiomyocytes were killed by T cell-directedcytolysis in response to the de-XTENylated HER2-PAT at approximate EC50concentration of 64 pM, while the cardiomyocytes remained refractory tokilling by XTENylated HER2-XPAT at concentrations as high as 1 μM,illustrating that the XTENylated PAT has less activity in thecardiomyocytes as compared to the cancer cell lines.

Example 10b

This example illustrates the sensitivity of another cell line withrelatively low level of HER2 expression, MCF-7, to T cell-directedcytotoxicity in response to increasing concentrations of a HER2-XPAT(e.g., set forth in Table D) and its active proteolytic product, thecorresponding HER2-PAT.

MCF-7 cells were cultured according to established protocols. Theconcentration-response curves and EC50 values were measured and analyzedaccording to the process outlined in Example 10a.

As shown in FIG. 6B, the MCF-7 cells were effectively killed by Tcell-directed cytolysis in response to the proteolytically de-XTENylatedHER2-XPAT (solid circles) at an EC50 concentration between 0.01 nM and0.1 nM. XTENylation on HER2-XPAT (e.g., set forth in Table D) reduced Tcell-mediated cytotoxicity by at least 10⁴-fold, as indicated by aright-ward shift of the dose-response curve (solid squares).

Example 10c

This example illustrates sensitivity of another cell line that expressesa medium level of HER2, MDA-MB-453, to T cell-directed cytotoxicity inresponse to increasing concentrations of a HER2-XPAT (e.g., set forth inTable D) and its active proteolytic product, the corresponding HER2-PAT.MDA-MB-453 is a breast cancer cell line with medium HER2 expression.

The cytotoxicity of an XTENylated PAT (as set forth in Table D) and thecorresponding de-XTENylated PAT (protease-treated) was determined usingan in vitro cytotoxicity assay which utilized the amount of ATP presentin wells of lysed target cells post treatment as a proxy for measuringcell viability. HER2-expressing target cells (MDA-MB-453 line) wereseeded on white, opaque bottom plates at 10k cells/well and allowed toincubate at 37° C., 5% CO₂ overnight (18-24 hours). Prior to the end ofthe overnight incubation, peripheral blood mononuclear cells (PBMCs)were thawed and incubated at 37° C., 5% CO₂ for 3-4 hours. The PBMCswere isolated from screened, healthy donors by Ficoll density gradientcentrifugation from either whole blood or from lymphocyte-enriched buffycoat preparations obtained from BioIVT. 10× XTENylated and de-XTENylatedPAT titrations were prepared using a 7-point, 5-fold titration with astarting concentration of 3000 nM for the XTENylated PAT and 10 nM forthe de-XTENylated PAT. The PBMCs were seeded in the wells at 10:1Effector: Target ratio. 10× XTENylated and de-XTENylated PAT titrationswere diluted 10-fold into the wells for starting concentrations of 300nM and 1 nM, respectively. The plates were incubated at 37° C., 5% CO₂for 48 hours. After the 48-hour incubation, the plates were washed 3×with 1×PBS, and 100 μL of 1×PBS was added to all wells. 100 μL ofCellTiter-Glo® luminescent substrate solution was added to all wells,and the plates were allowed to incubate at room temperature for 1-5minute(s). The plates were then shaken on a plate shaker at 300-500 rpmfor 30-60 seconds to mix the contents of the wells and then read in aluminometer using an integration time of 100 ms. The intensity of signalproduced correlates to the amount of viable cells present in the wells.The mean of the signal from all non-treatment wells was calculated andused to determine % Live cells from treatment wells ((TreatmentSignal/Mean of Non-Treatment Signal)*100=% Live). The % Live was plottedby concentration, and half maximal response (EC50) values were derivedwith a 4-parameter logistic regression equation using GraphPad Prismsoftware.

The concentration-response curves for the de-XTENylated HER2-XPAT (solidcircles) and the XTENylated HER2-PAT (solid squares) are shown,respectively, in FIG. 6C.

HER2 expression levels of the cell lines tested herein are known in theart. For example, Hendriks et al. (Mol. Cancer Ther. Sep. 1, 2013;12(9):1816-1828), which is incorporated by reference herein in itsentirety, details mean HER2 level of various cells in FIG. 1E (X-axisindicates number of HER2 receptors per cell). BT-474 can have arelatively high HER2 level (an immunohistochemistry test score) of “3+”;cardiomyocytes can have a HER2 level of “2+”; and MCF-7 can have arelatively low HER2 level of “1+/0.”

Example 11. Tumor Regression Induced in Subjects by Administration ofXTENylated XPATs

This example illustrates tumor regression induced in subjects byadministration of vehicle, non-cleavable XTENylated HER2-PAT, cleavableXTENylated HER2-PAT, and unmasked HER2-PAT. The non-cleavable constructsused in the experiments are identical to the corresponding cleavableconstructs, but the release site has been replaced with a non-cleavablesequence of similar length made from GASTEP amino acids (glycine,alanine, serine, threonine, glutamate, and/or proline).

Example 11a

Mice were implanted with estrogen pellets (17β-estradiol, 60 dayrelease) at the right flank one day before the tumor inoculation. BT-474tumor cells were inoculated subcutaneously at the right flank region ofeach mouse at a concentration of 2×10⁷ BT-474/200 ul RPMI 1640 withMatrigel (1:1)/mouse for tumor development. The date of tumor cellinoculation was denoted as Day 0 (D0).

When tumors become palpable (mean tumor volume (MTV)=117 mm³) on D8,PBMCs were intravenous implanted at 1×10⁷ PBMC/200 ul RPMI 1640/mouse.One group of mice was not injected with PBMCs (the “without hPBMCs”group) and underwent the remainder of experimental treatments inparallel with the PBMC-injected mice. When mean tumor volume reached 147mm³ (D10), the “without hPBMCs” group of mice was administered withvehicle (diluent) (solid square in FIG. 7A); and the PBMC-injected micewere randomized into four groups and administered with vehicle (diluent)(solid circle in FIG. 7A), unmasked bispecific (solid triangle in FIG.7A), cleavable XTENylated bispecific (hexagon in FIG. 7A), andnon-cleavable XTENylated bispecific (diamond in FIG. 7A), respectively.All five groups received an equimolar dose at 15 nmol/Kg.

As illustrated in FIG. 7A, by the end of study, D35, the cleavableXTENylated HER2-PAT (e.g., set forth in Table D) and the unmaskedbispecific both demonstrated significant anti-tumor efficacy whencompared to controls (vehicle with or without hPBMC) and thenon-cleavable XTENylated bispecific counterpart. The lack of any tumorgrowth inhibition of the non-cleavable XTENylated bispecific constructsupports that the anti-tumor efficacy observed on the XTENylated HER2bispecific is driven by proteolytic cleavage.

Example 11b

The experimental setup implemented was similar to that described inExample 11a. Mice were implanted with estrogen pellets (17β-estradiol,60 day release) at the right flank one day before the tumor inoculation.BT-474 tumor cells were inoculated subcutaneously at the right flankregion of each mouse at a concentration of 2×10⁷ BT-474/200 ul RPMI 1640with Matrigel (1:1)/mouse for tumor development. The date of tumor cellinoculation was denoted as Day 0 (DO). When tumors become palpable(MTV=94 mm³) on D8, PBMCs were intravenous implanted at 1×10⁷ PBMC/200ul RPMI 1640/mouse. One group of mice was not injected with PBMCs (the“without hPBMCs” group) and underwent the remainder of experimentaltreatments in parallel with the PBMC-injected mice. When MTV reached 444mm³ (D20), the PBMC injected mice were randomized, and vehicle (diluent)and test article treatments were delivered as a single dose for allgroups on D21.

FIG. 7B illustrates significant anti-tumor efficacy of cleavableXTENylated HER2-XPAT (e.g., set forth in Table D) even in largeestablished tumors (e.g., mean tumor volume (MTV)>400 mm³) after asingle dose, when compared to controls and the non-cleavable XTENylatedbispecific counterpart. The lack of appreciable level of tumor growthinhibition achieved by the non-cleavable XTENylated HER2-PAT supportsthat the anti-tumor efficacy of the cleavable XTENylated HER2-PAT isdriven by proteolytic cleavage.

Example 11c

Examples 11a and 11b used xenografts generated from a breast cancer cellline that expresses relatively high levels of HER2, in the presentexample, the tumor regression studies were performed using a colorectalxenograft model that is characterized by low levels of HER2 expression.Mice were inoculated subcutaneously at the right flank region of eachmouse at a concentration of 5×10⁶ HT-55/200 ul RPMI 1640 with Matrigel(1:1)/mouse for tumor development. The date of tumor cell inoculationwas denoted as Day 0 (D0).

When tumors become palpable (mean tumor volume (MTV)=82 mm³) on D6,PBMCs were intravenously implanted at 1×10⁷ PBMC/200 ul RPMI 1640/mouse.One group of mice was not injected with PBMCs (the “without hPBMCs”group) and underwent the remainder of experimental treatments inparallel with the PBMC-injected mice. When mean tumor volume reached 130mm³ (D10), the “without hPBMCs” group of mice was administered withvehicle (diluent) (solid square in FIG. 7C); and the PBMC-injected micewere randomized into five groups and administered with vehicle (diluent)(solid circle in FIG. 7C), unmasked bispecific at 15 nmol/Kg (solidtriangle in FIG. 7C), cleavable XTENylated bispecific at 15 nmol/Kg(triangle in FIG. 7C) cleavable XTENylated bispecific at 36 nmol/Kg(hexagon in FIG. 7C), and non-cleavable XTENylated bispecific. (diamondin FIG. 7A)

As illustrated in FIG. 7C, by the end of the study the cleavableXTENylated HER2-PAT (e.g., set forth in Table D) and the unmaskedbispecific (at both 15 nmol/Kg and 36 nmol/Kg) demonstrated significantanti-tumor efficacy when compared to controls (vehicle with or withouthPBMC) and the non-cleavable XTENylated bispecific counterpart. The lackof any tumor growth inhibition of the non-cleavable XTENylatedbispecific construct supports that the anti-tumor efficacy observed onthe XTENylated HER2 bispecific is driven by proteolytic cleavage.

In additional in vivo experiments, it was demonstrated that HER2-XPAT(i.e., XTENylated or masked HER2-PAT) is preferentially unmasked intumor tissue with significantly greater cleavage seen in tumors ascompared to normal tissue. BT-474 tumors were established in NSG mice.The mice were injected with HER2-XPAT that was site-specificallylabelled with an IR dye. After a period of 2 days, the tissue (tumor,brain, heart and liver) was harvested, homogenized and the presence ofunmasked HER2-PAT in the various tissues of the BT-474 was assessed.Further, in order to determine whether the cleavage/unmasking of theHER2-XPAT occurred in vivo in the tumor or whether it was an artefact ofthe homogenization of the tissue once the tissue had been harvested, thesamples were spiked with a differently labeled HER2-XPAT prior to samepreparation. FIG. 7D demonstrates that there is preferential cleavage ofHER2-XPAT to the unmasked PAT in the tumor tissue as compared to thecleavage seen in the brain, heart, and liver tissue. Further, FIG. 7Dalso shows that the cleavage occurred in vivo in the tissue as opposedto ex vivo as a result of homogenization or other handling. In addition,there is strong evidence of XPAT cleavage in fresh tumor samples.

Examples 11a-11c demonstrate that cleavable XTENylated HER2-PAT inducesrobust tumor regression in tumor models that express low levels of HER2as well as tumors that are characterized with high HER2 expression. Assuch tumor regression is not observed in the test subjects treated withnon-cleavable XPAT. Moreover, the data presented herein show thatHER2-XPAT is preferentially unmasked in vivo, in the tumormicroenvironment. The experimental design for Examples 11a-11c arefurther discussed below in Example 17.

Example 12. Lymphocyte Margination Induced in Subjects by Administrationof XTENylated XPATs

This example illustrates lymphocyte margination induced in subjects(e.g., female and male monkeys) by administration of an XTENylatedHER2-PAT (e.g., as set forth in Table 6), for example, a single-doseintravenous (IV) infusion (e.g., at 25 mg/kg at a dose volume of 10ml/kg).

Test article dosing formulations were prepared by diluting theXPAT-of-interest according to established protocols to meet dose levelrequirements. The test article (the HER2-XPAT of interest) and theappropriate diluent were provided as single-use aliquots and stored in afreezer set to maintain −80° C. until use. Once thawed, aliquots werestored at 2° C.-8° C. and were not refrozen. The dosing formulationswere prepared on the day of dosing. On the day of use, an aliquot ofXPAT stock solution and the appropriate diluent was thawed at roomtemperature and an appropriate amount of test article was diluted toobtain the proper concentration. The dosing formulation was mixed byinverting the tube gently several times. The IV route of exposure wasselected as a candidate route of human exposure.

As shown in FIG. 8 , the HER2-XPAT-related changes in hematologyparameters following a single dose (25 mg/kg), including a decrease oflymphocytes (summarized in the table immediately below), were observedstarting at 6 hours post-dose (0.5-0.2× of the pre-dose level) andsustained at 24 hours post-dose (0.27-0.1× of the pre-dose level) and at48 h (0.09-0.36× of the pre-dose level). At 72 hr post-dose, thelymphocyte level remained low (0.3-0.1× of the pre-dose level).

TABLE 14 Hematology: lymphocyte changes associated with HER2-XPATadministration Dose (25 mg/kg) Parameter Time Point M F LymphocytePre-dose 7.22 7.31 count  6 hours post-dose 1.49 3.87 24 hours post-dose0.7 1.98 48 hours post-dose 0.67 2.63 72 hours post-dose 0.84 2.27

Example 13. Evaluation of Toxicity and Pharmacokinetics of XTENylatedPATs (XPATs) in Subjects

Toxicity and pharmacokinetics of XTENylated HER2-PAT (e.g., set forth inTable D) were assessed in cynomolgus monkeys. Animals were dosed with 25mg/kg of a cleavable XTENylated HER2-PAT or a correspondingnon-cleavable XTENylated counterpart over 1 hour. Blood for plasma wascollected at pre-dose and post end of infusion at: 5 min, 2 hrs, 4 hrs,6 hrs, 24 hrs, 48 hrs, 96 hrs, 144 hrs, 168 hrs, and 240 hrs. All bloodwas collected on time+/−2 minutes. Plasma samples were analyzed for theconcentration of the cleavable and non-cleavable XTENylated bispecificconstructs. The plasma drug concentrations were measured by ECLIA(electrochemiluminescent immunoassay) using recombinant HER2 as captureand an antibody directed against the XTEN mask for detection. As shownin FIG. 9 , in cynomolgus monkeys, the HER2-XPAT (e.g., set forth inTable D) showed a pharmacokinetic profile analogous to the correspondingnon-cleavable version due to its stability in peripheral tissues. Thenon-cleavable constructs used in the experiments are identical to thecorresponding cleavable constructs, but for the fact that the releasesite has been replaced with a non-cleavable sequence of similar lengthmade from GASTEP amino acids (glycine, alanine, serine, threonine,glutamate, and/or proline). The presence of proteolytic metabolites incirculation after two separate doses was also monitored and shown inFIG. 9B as a percentage of the total HER2-XPAT. As shown in FIG. 9B thepresence of the 1x-metabolites is less than 4% of the total HER2-XPAT.

Example 14. XTEN Masking Expands Safety Margin of HER2-XPAT Vs. HER2-PAT

Additional data shows that HER2-XPAT is well-tolerated in cynomolgusmonkeys and the presence of the provides a 450-fold higher toleratedCmax as compared to that seen with unmasked PAT. HER2-XPAT wasadministered IV, single dose/animal (doses 2.5-42 mpk) and weekly ×2 at50 mpk. *At doses 21 mpk and above, a variant of HER2-XPAT with ashorter C-terminal XTEN mask was used. HER2-PAT was administered bycontinuous infusion due to its short half-life. Plasma concentrations ofHER2-XPAT were measured by ECLIA* using recombinant HER2 capture and anantibody directed against the XTEN mask for detection. The Cmax valuesfor HER2 PAT were determined by ECLIA utilizing an a-idiotypic Abdirected against the a-CD3 scFv as capture and recombinant HER2 asdetection. *ECLIA=Electrochemiluminescent Immunoassay. FIG. 10A showsthe maximum tolerated dose of 42 mg/kg, indeed no overt CRS was seeneven at 50 mg/kg. Conversely, in FIG. 10B the maximum tolerated dose ofthe unmasked HER2-PAT is 0.2 mg/kg. FIG. 10C shows the plasmaconcentrations of masked HER2-PAT (at a 450-fold higher tolerated Cmax)as compared to unmasked HER2-PAT.

In FIG. 13A-FIG. 13B there is additional evidence supporting the safetyprofile of a preferred HER2-XPAT of the present invention. HER2-XPAT orits non-cleavable counterpart, HER2-XPAT-NoClvSite were administered asa single IV dose of 25 or 42 mg/kg. Dose normalized plasma drugconcentrations were measured by ECLIA* using recombinant HER2 as captureand an antibody directed against the XTEN mask for detection.*ECLIA=Electrochemiluminescent Immunoassay. The data in FIG. 12A shows acomparable PK between HER2-XPAT and the non-cleavable HER2-PAT formats,demonstrating that the protease release site remains largely stable incirculation of cynomolgus monkeys even at high doses. In FIG. 12B,concentrations of singly-cleaved HER2-XPAT(1X-C) and HER2-XPAT(1X-N)were measured in plasma from cynomolgus monkeys administered HER2-XPAT.Quantitative Western was performed using an antibody recognizing theanti-HER2 scFv with standards prepared with recombinant versions of thesingly-cleaved molecules. Results are expressed as a percentage of thetotal XTENylated species as measured by ECLIA as described above. FIG.12B shows that even at high dose of HER2 XPAT there is very limitedsystemic accumulation of metabolites lacking one or both XTEN masks.

Example 15. HER2-XPAT does not Induce Cytokine Release Syndrome

Consistent with the tolerability of HER2-XPAT, the present Exampledemonstrates that HER2-XPAT in pilot NHP toxicology studies led tominimal T cell activation in circulation in NHPs, while its unmaskedcounterpart led to significant T cell activation even at much lowerdoses. Consistent with this masking of T cell activation, cytokinerelease upon administration of HER2-XPAT was also robustly attenuatedrelative to what was observed with its unmasked counterpart. There wasno overt Cytokine Release Syndrome (CRS) with HER2-XPAT, even at the MTDof 42 mg/kg, while its unmasked TCE counterpart caused death due to CRSat doses as low as 0.3 mg/kg. Overall, AMX-818 was well-tolerated in NHPstudies at doses that would be significantly higher than anticipatedclinically relevant doses, and there were no gross or microscopicfindings in HER2-expressing tissues such as the heart, where otherapproved HER2-targeted agents have demonstrated clinical toxicity.

Thus, even at 50 mg/kg, administration of HER2-XPAT did not inducecytokine release or systemic T cell activation. This supports that thereis minimal CRS risk for XPATs vs standard TCEs. Only 1-3% ofsingly-cleaved XPAT metabolites were detected in plasma from NHPadministered high doses of HER2-XPAT (25 & 42 mg/kg). For this study,peripheral T cell activation (% CD25⁺) was evaluated by flow cytometry24 hours post-HER2-XPAT treatment. FIGS. 11A and 11B show CD25⁺CD4⁺ Tcell (FIG. 11A) and CD25⁺CD8⁺ (FIG. 11B) T cell in the presence ofHER2-XPAT vs. HER2-PAT.

Cytokine analysis was performed with a Luminex® suspension array systemon plasma samples. FIGS. 11C-11E show the IL-6 (FIG. 11C), TNF-a (FIG.11D) and IFN-g (FIG. 11E) levels in plasma in response to HER2-XPAT vsHER2-PAT. Data presented are maximal values measured between 6-24 hoursat each evaluated dose. In additional GLP toxicology studies (data notshown), NHPs were dosed weekly with AMX-818 at 3 dose levels (0.5 mg/kg,2 mg/kg and 6 mg/kg) over a 31-day period. The results from these GLPtoxicology studies are consistent with the prior pilot NHP studies andthere were no AMX-818 related adverse findings based on standardtoxicology endpoints, including histopathologic examination, and allclinical pathological findings were reversible.

Also consistent with the safety of AMX-818 in NHPs up to 42 mg/kg,AMX-818 is largely stable in circulation where protease inhibitors areabundant, as evidenced by the limited systemic accumulation of activemetabolites. Because healthy NHPs were dosed and subjects to be treatedwith AMX-818 also may have systemic inflammation, the stability ofAMX-818 was analyzed ex vivo in plasma samples taken from patients withcancer and inflammatory disease, as well as NHPs in which we inducedsystemic inflammation. As shown in FIG. 12 , similar AMX-818 stabilitywas observed in plasma samples from healthy NHPs, inflamed NHPs, healthyhumans, and human patients with cancer or inflammatory disease in astability study in which we incubated AMX-818 with plasma samples forseven days at 37° C.

For this experiment, AMX-818 with a fluorescent label (DyL650) attachedto the tandem scFv was incubated in the indicated plasma samples forseven days at 37° C. Samples were then run on a gel and metabolitessimilar in size to the unmasked, active form of AMX-818 were quantifiedusing a LI-COR detector. Inflammatory disease human samples were derivedfrom patients with rheumatoid arthritis, lupus, inflammatory boweldisease, and multiple sclerosis. Cancer human samples were derived frompatients with lung, breast, and colon tumors. Sample sizes: Healthy NHPN=4, Healthy Human N=4, “Inflamed” NHP N=6, “Inflamed” Human N=27,Cancer Human N=11.

Across all four plasma groups, the range of products similar in size tothe active, unmasked HER2 TCE was only approximately two to four percentof the total AMX-818 species present. This small percentage reflects anoverestimate of potential accumulation of unmasked TCE in vivo, as renalfiltration will rapidly eliminate such products from circulation(half-life of the unmasked form in NHP is approximately seven hours).These data suggest limited risk of significant systemic accumulation ofthe active, unmasked form of AMX-818 in cancer patients.

Example 16. AMX-818 and 818-PAT Induce Surface Expression of PD-1 andCD69 on T Cells in Response to SKOV3 Tumor Cells

Surface PD-1 expression was evaluated on CD4+ and CD8+ T cells by flowcytometry following a 48 hour co-incubation of PBMCs and SKOV3 cells ata 5:1 Effector:Target ratio with test articles at the indicatedconcentrations. On both CD4 and CD8 T cell subsets, 818-PAT and AMX-818increased the frequency of T cells expressing surface PD-1, with theAMX-818 curve shifted approximately 2.5-logs relative to 818-PAT,demonstrating significant masking by the prodrug relative to itsactivated counterpart. Maximal activation of the T cells by AMX-818(PAT)was achieved at 625 μM concentrations, while maximal activation byAMX-818 was achieved at 150-600 nM concentrations.

Histogram plots comparing the surface levels of PD-1 on CD4 and CD8 Tcells at the indicated drug concentrations are shown in FIG. 14A-14D.

AMX-818(PAT) and prodrug AMX-818 induced CD69 and PD-1 expression on thesurface both CD4+ and CD8+ T-cell subsets to comparable degrees.However, the dose-response curve for AMX-818 was shifted onaverage >400-fold higher relative to that of the activated AMX-818(PAT),demonstrating effective functional masking of AMX-818 (data shown forCD8 T cells in FIG. 14A and FIG. 14C). Maximal upregulation ofactivation marker CD69 and inhibitory receptor PD-1 by AMX-818(PAT) wasobserved at 0.62 nM concentrations, while comparable induction byAMX-818 required 150-600 nM concentrations (FIGS. 14B and 14D).

AMX-818(PAT) also potently induced surface expression of PD-L1 on SKOV3tumor cells with an EC₅₀ value of 19.7 pM, while XTENylated AMX-818 was650-fold less potent in inducing comparable frequency and levels ofPD-L1 (see FIG. 15A-15C). The induction of PD-1 on T cells and PD-L1 ontarget tumor cells by AMX-818 is expected to dampen its activity andprovides rationale for combining a PD-1 blocking therapy to AMX-818 infuture clinical investigations.

AMX-818(PAT) and AMX-818 induced PD-1 to comparable levels and maximalpercentages within both human CD4⁺ and CD8⁺ T-cell subsets. However,AMX-818 was on average >400-fold less potent than AMX-818(PAT).AMX-818(PAT) potently induced surface expression of PD-L1 on HER2-highexpressing SKOV3 tumor cells with an EC₅₀ value of 19.7 pM, whileAMX-818 was >650-fold less potent for comparable induction. Theinduction of PD-1 on T cells and PD-L1 on target tumor cells by AMX-818is expected to dampen its activity and provides the rationale for acombination of PD-1 blocking therapy with AMX-818.

Example 17. XPATs are Unmasked to TCEs in Human Tumors Implanted inLiving Mice, with Minimal Cleavage Observed in Healthy Tissues

The present example summarizes the potent in vivo efficacy of theHER2-XPATS of the present invention. To assess anti-tumor activity,immunocompromised mice were injected with tumor cells expressing thetarget of interest. When the tumors were sufficiently large andwell-established (over 100 mm³), human PBMCs were injected andintravenous dosing of the HER2-XPATs and a variety of control moleculeswas initiated. These studies showed that the HER2 targeting XPATs drovetumor regressions and a number of complete responses. An AMX-818 variantlacking the protease-cleavable linker had no activity, indicating thatprotease cleavage of the linker drove activity.

Moreover, the HER2-XPAT expand the safety margin in vivo. HER2 hasrelatively limited expression in healthy tissues. When NHPs wereinjected with the HER2-XPATs, as well as their unmasked TCEcounterparts, and tolerability was assessed to determine the maximumtolerated dose of each molecule. XTEN masks on the XPATs significantlyincreased the tolerability of the TCEs, enabling an over 400-fold higherCmax at MTD relative to what is observed with the unmasked HER2 TCE. Toprovide evidence that the protease-cleavable linker can be cleavedacross a broad range of human tumors, XPAT cleavage was analyzed infresh human tumor samples, as well as patient-derived and xenografttumors. In fresh human tumors explants, an XPAT inducedprotease-cleavable linker-dependent release of cytokines from tumorresident T cells, similar to the cytokine release induced by a positivecontrol (anti-CD3 antibody) that directly activated T cells.

To quantify the degree of preferential unmasking of XPATs that occurs intumors, XPAT cleavage was assessed in living mice. A fluorophore labeledXPAT was injected into mice, and two days later harvested tumors andhealthy tissues to measure levels of XPAT and cleavage products. Tospecifically quantify cleavage occurring in the living mouse, andcontrol for any cleavage occurring after tissues were harvested, we alsoanalyzed cleavage of a control XPAT added during tissue processing.Across 31 mice with nine different tumor types, on average, 20% of theXPAT in tumors of living mice was cleaved to the unmasked TCE form,while minimal cleavage of XPATs was observed in healthy tissues. Thedemonstration that there was limited-to-no cleavage of the control XPATconfirms that cleavage of the injected XPAT occurred in the living mice,not during tissue processing. The study design and data are shown inFIG. 16A-C.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. It is not intendedthat the invention be limited by the specific examples provided withinthe specification. While the invention has been described with referenceto the aforementioned specification, the descriptions and illustrationsof the embodiments herein are not meant to be construed in a limitingsense. Numerous variations, changes, and substitutions will now occur tothose skilled in the art without departing from the invention.Furthermore, it shall be understood that all aspects of the inventionare not limited to the specific depictions, configurations or relativeproportions set forth herein which depend upon a variety of conditionsand variables. It should be understood that various alternatives to theembodiments of the invention described herein may be employed inpracticing the invention. It is therefore contemplated that theinvention shall also cover any such alternatives, modifications,variations or equivalents. It is intended that the following claimsdefine the scope of the invention and that methods and structures withinthe scope of these claims and their equivalents be covered thereby.

1. A polypeptide having an N-terminal amino acid and a C-terminal aminoacid, the polypeptide comprising: (a) an extended recombinantpolypeptide (XTEN), comprising: a barcode fragment (BAR) releasable fromsaid polypeptide upon digestion by a protease; (b) a bispecific antibodyconstruct (BsAb), comprising: a first antigen binding fragment (AF1)that specifically binds to cluster of differentiation 3 T cell receptor(CD3), which AF1 comprises light chain complementarity-determiningregions 1 (CDR-L1), 2 (CDR-L2), and 3 (CDR-L3) and heavy chaincomplementarity-determining regions 1 (CDR-H1), 2 (CDR-H2), and 3(CDR-H3), wherein said CDR-H3 comprises an amino acid sequence of SEQ IDNO:10; and a second antigen binding fragment (AF2) that specificallybinds to human epidermal growth factor receptor 2 (HER2); and (c) arelease segment (RS) positioned between said XTEN and said bispecificantibody construct, wherein said XTEN is characterized in that: (i) itcomprises at least 100, or at least 150 amino acids; (ii) at least 90%of its amino acid residues are identified herein by glycine (G), alanine(A), serine (S), threonine (T), glutamate (E) or proline (P); (iii) itcomprises at least 4 different types of amino acids identified herein byG, A, S, T, E, or P; and (iv) said XTEN is formed from a plurality ofnon-overlapping sequence motifs that are each from 9 to 14 amino acidsin length, wherein said plurality of non-overlapping sequence motifscomprise: (1) a set of non-overlapping sequence motifs, wherein eachnon-overlapping sequence motif of said set of non-overlapping sequencemotifs is repeated at least two times in said XTEN; and (2) anon-overlapping sequence motif that occurs only once within said XTEN;wherein said barcode fragment (BAR) includes at least part of saidnon-overlapping sequence motif that occurs only once within said XTEN;wherein said barcode fragment (BAR) differs in sequence and molecularweight from all other peptides fragments that are releasable from saidpolypeptide upon complete digestion of said polypeptide by saidprotease; and wherein said barcode fragment (BAR) does not include saidN-terminal amino acid or said C-terminal amino acid of said polypeptide.2. The polypeptide of claim 1, wherein: a. said set of non-overlappingsequence motifs are each independently identified herein by SEQ ID NOS:179-200 and 1715-1722; b. said set of non-overlapping sequence motifsare each independently identified herein by SEQ ID NOS: 186-189; c. saidset of non-overlapping sequence motifs comprise at least two, at leastthree, or all four of the sequence motifs SEQ ID NOS: 186-189. 3.(canceled)
 4. (canceled)
 5. The polypeptide of claim 1, wherein: a. saidXTEN comprises a length of from 100 to 3,000, from 150 to 3,000, from100 to 1,000, or from 150 to 1,000 amino acid residues; b. said XTENcomprises a length of at least 200, at least 250, at least 300, at least350, at least 400, at least 450, or at least 500 amino acid residues; c.at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of theamino acid residues of said XTEN are glycine (G), alanine (A), serine(S), threonine (T), glutamate (E) or proline (P); and/or d. said XTENhas at least 90%, at least 92%, at least 95%, at least 98%, at least 99%or 100% sequence identity to a sequence set forth in Table 3a. 6-8.(canceled)
 9. The polypeptide of claim 1, wherein said barbode fragment(BAR): a. does not include a glutamic acid that is immediately adjacentto another glutamic acid, if present, in said XTEN; b. has a glutamicacid at its C-terminus; c. has an N-terminal amino acid that isimmediately preceded by a glutamic acid residue; d. is positioned adistance from either said N-terminus of said polypeptide or saidC-terminus of said polypeptide, wherein said distance is from 10 to 150amino acids, or from 10 to 125 amino acids in length; e. ischaracterized in that: i. it does not include a glutamic acid that isimmediately adjacent to another glutamic acid, if present, in said XTEN;ii. it has a glutamic acid at its C-terminus; iii. it has an N-terminalamino acid that is immediately preceded by a glutamic acid residue; andiv. it is positioned a distance from either said N-terminus of saidpolypeptide or said C-terminus of said polypeptide, wherein saiddistance is from 10 to 150 amino acids, or from 10 to 125 amino acids inlength; optionally wherein said glutamic acid residue that precedes saidN-terminal amino acid of said barcode fragment (BAR) is not immediatelyadjacent to another glutamic acid residue; and/or f. does not include asecond glutamic acid residue at a position other than the C-terminus ofsaid barcode fragment unless said second glutamic acid is immediatelyfollowed by a proline. 10-15. (canceled)
 16. The polypeptide of claim 1,wherein said XTEN is positioned: a. N-terminal of said bispecificantibody construct (BsAb), and wherein said barcode fragment (BAR) ispositioned within 200, within 150, within 100, or within 50 amino acidsof said N-terminus of said polypeptide; b. N-terminal of said bispecificantibody construct (BsAb), and wherein said barcode fragment (BAR1) ispositioned at a location that is between 10 and 200, between 30 and 200,between 40 and 150, or between 50 and 100 amino acids from saidN-terminus of said protein; c. C-terminal of said bispecific antibodyconstruct (BsAb), and said barcode fragment (BAR) is positioned at alocation that is between 10 and 200, between 30 and 200, between 40 and150, or between 50 and 100 amino acids from said C-terminus of saidprotein; and/or d. C-terminal of said bispecific antibody construct(BsAb), and said barcode fragment (BAR) is positioned at a location thatis between 10 and 200, between 30 and 200, between 40 and 150, orbetween 50 and 100 amino acids from said C-terminus of said protein.17-19. (canceled)
 20. The polypeptide of claim 1, wherein: a. saidbarcode fragment (BAR) is at least 4 amino acids in length; b. saidbarcode fragment (BAR) is between 4 and 20, between 5 and 15, between 6and 12, or between 7 and 10 amino acids in length; c. said barcodefragment (BAR) comprises an amino acid sequence set forth in Table 2; d.said XTEN has a length defined by a proximal end and a distal end,wherein (1) said proximal end is positioned, relative to said distalend, closer to said bispecific antibody construct (BsAb), and wherein(2) said barcode fragment (BAR) is positioned within a region of saidXTEN that extends, as measured from said distal end, between 5% and 50%,between 7% and 40%, or between 10% and 30% of said length of said XTEN;e. said XTEN further comprises additional one or more barcode fragments,wherein said additional one or more barcode fragments each differ insequence and molecular weight from all other peptides fragments that arereleasable from said polypeptide upon complete digestion of saidpolypeptide by said protease; f. said release segment (RS) comprises anamino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or 100% sequence identity to a sequence identified hereinby SEQ ID NOS: 7001-7626; g. said protease cleaves on the C-terminalside of glutamic acid residues that are not followed by proline,optionally wherein said protease is a Glu-C protease; and/or h. thepolypeptide is expressed as a fusion protein, wherein said fusionprotein, in an uncleaved state, has a structural arrangement fromN-terminus to C-terminus identified herein by AF1-AF2-RS-XTEN,AF2-AF1-RS-XTEN, XTEN-RS-AF1-AF2, or XTEN-RS-AF2-AF1. 21-28. (canceled)29. The polypeptide of claim 1, wherein said release segment (RS) isfused to said bispecific antibody construct (BsAb) by a spacer,optionally wherein: a. said spacer comprises at least 4 types of aminoacids are glycine (G), alanine (A), serine (S), threonine (T), glutamate(E) or proline (P); or b. said spacer comprises an amino acid sequencehaving at least 80%, 90%, or 100% sequence identity to a sequence setforth in Table C.
 30. (canceled)
 31. (canceled)
 32. The polypeptide ofclaim 1, wherein said CDR-H1 and said CDR-H2 of said first antigenbinding fragment (AF1) comprise amino acid sequences of SEQ ID NOS: 8and 9, respectively; and/or a. said CDR-L1 of said AF1 comprises anamino acid sequence of SEQ ID NO:1 or 2; said CDR-L2 of said AF1comprises an amino acid sequence of SEQ ID NO:4 or 5; and said CDR-L3 ofsaid AF1 comprises an amino acid sequence of SEQ ID NO:6; b. said CDR-L1of said AF1 comprises an amino acid sequence of SEQ ID NO:1; said CDR-L2of said AF1 comprises an amino acid sequence of SEQ ID NO:4 or 5; andsaid CDR-L3 of said AF1 comprises an amino acid sequence of SEQ ID NO:6;c. said CDR-L1 of said AF1 comprises an amino acid sequence of SEQ IDNO:2; said CDR-L2 of said AF1 comprises an amino acid sequence of SEQ IDNO:4 or 5; and said CDR-L3 of said AF1 comprises an amino acid sequenceof SEQ ID NO:6; d. said CDR-L1 of said AF1 comprises an amino acidsequence of SEQ ID NO:1; said CDR-L2 of said AF1 comprises an amino acidsequence of SEQ ID NO:4; and said CDR-L3 of said AF1 comprises an aminoacid sequence of SEQ ID NO:6; or e. said CDR-L1 of said AF1 comprises anamino acid sequence of SEQ ID NO:2; said CDR-L2 of said AF1 comprises anamino acid sequence of SEQ ID NO:5; and said CDR-L3 of said AF1comprises an amino acid sequence of SEQ ID NO:6. 33-37. (canceled) 38.The polypeptide of claim 1, wherein said first antigen binding fragment(AF1): a. comprises four chain variable domain framework regions 1(FR-H1), 2 (FR-H2), 3 (FR-H3), and 4 (FR-H4), each exhibiting at least86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity or being identical to an amino acid sequence of: i.SEQ ID NOS: 60, 64, 65, and 67, respectively; or ii. SEQ ID NOS: 61, 64,65, and 67, respectively; b. comprises four light chain variable domainframework regions (FR-L): FR-L1, FR-L2, FR-L3, and FR-L4, eachexhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% sequence identity or being identical to amino acidsequences of: i. SEQ ID NOS: 51, 52, 53, and 59, respectively; ii. SEQID NOS: 51, 52, 54, and 59, respectively; iii. SEQ ID NOS: 51, 52, 55,and 59, respectively; iv. SEQ ID NOS: 51, 52, 56, and 59, respectively;and/or c. further comprises: i. light chain framework regions 1 (FR-L1),2 (FR-L2), 3 (FR-L3), and 4 (FR-L4) and heavy chain framework regions 1(FR-H1), 2 (FR-H2), 3 (FR-H3) and 4 (FR-H4), wherein: said FR-L1comprises an amino acid sequence of SEQ ID NO:51; said FR-L2 comprisesan amino acid sequence of SEQ ID NO:52; said FR-L3 comprises an aminoacid sequence of SEQ ID NO:53, 54, 55, or 56; said FR-L4 comprises anamino acid sequence of SEQ ID NO:59; said FR-H1 comprises an amino acidsequence of SEQ ID NO:60 or 61; said FR-H2 comprises an amino acidsequence of SEQ ID NO:64; said FR-H3 comprises an amino acid sequence ofSEQ ID NO:65; and said FR-H4 comprises an amino acid sequence of SEQ IDNO:67; ii. light chain framework regions 1 (FR-L1), 2 (FR-L2), 3(FR-L3), and 4 (FR-L4) and heavy chain framework regions 1 (FR-H1), 2(FR-H2), 3 (FR-H3) and 4 (FR-H4), wherein: said FR-L1 comprises an aminoacid sequence of SEQ ID NO:51; said FR-L2 comprises an amino acidsequence of SEQ ID NO:52; said FR-L3 comprises an amino acid sequence ofSEQ ID NO:53; said FR-L4 comprises an amino acid sequence of SEQ IDNO:59; said FR-H1 comprises an amino acid sequence of SEQ ID NO:60; saidFR-H2 comprises an amino acid sequence of SEQ ID NO:64; said FR-H3comprises an amino acid sequence of SEQ ID NO:65; and said FR-H4comprises an amino acid sequence of SEQ ID NO:67; iii. light chainframework regions 1 (FR-L1), 2 (FR-L2), 3 (FR-L3), and 4 (FR-L4) andheavy chain framework regions 1 (FR-H1), 2 (FR-H2), 3 (FR-H3) and 4(FR-H4), wherein: said FR-L1 comprises an amino acid sequence of SEQ IDNO:51; said FR-L2 comprises an amino acid sequence of SEQ ID NO:52; saidFR-L3 comprises an amino acid sequence of SEQ ID NO:54; said FR-L4comprises an amino acid sequence of SEQ ID NO:59; said FR-H1 comprisesan amino acid sequence of SEQ ID NO:61; said FR-H2 comprises an aminoacid sequence of SEQ ID NO:64; said FR-H3 comprises an amino acidsequence of SEQ ID NO:65; and said FR-H4 comprises an amino acidsequence of SEQ ID NO:67; iv. light chain framework regions 1 (FR-L1), 2(FR-L2), 3 (FR-L3), and 4 (FR-L4) and heavy chain framework regions 1(FR-H1), 2 (FR-H2), 3 (FR-H3) and 4 (FR-H4), wherein: said FR-L1comprises an amino acid sequence of SEQ ID NO:51; said FR-L2 comprisesan amino acid sequence of SEQ ID NO:52; said FR-L3 comprises an aminoacid sequence of SEQ ID NO:55; said FR-L4 comprises an amino acidsequence of SEQ ID NO:59; said FR-H1 comprises an amino acid sequence ofSEQ ID NO:61; said FR-H2 comprises an amino acid sequence of SEQ IDNO:64; said FR-H3 comprises an amino acid sequence of SEQ ID NO:65; andsaid FR-H4 comprises an amino acid sequence of SEQ ID NO:67; v. lightchain framework regions 1 (FR-L1), 2 (FR-L2), 3 (FR-L3), and 4 (FR-L4)and heavy chain framework regions 1 (FR-H1), 2 (FR-H2), 3 (FR-H3) and 4(FR-H4), wherein: said FR-L1 comprises an amino acid sequence of SEQ IDNO:51; said FR-L2 comprises an amino acid sequence of SEQ ID NO:52; saidFR-L3 comprises an amino acid sequence of SEQ ID NO:56; said FR-L4comprises an amino acid sequence of SEQ ID NO:59; said FR-H1 comprisesan amino acid sequence of SEQ ID NO:61; said FR-H2 comprises an aminoacid sequence of SEQ ID NO:64; said FR-H3 comprises an amino acidsequence of SEQ ID NO:65; and said FR-H4 comprises an amino acidsequence of SEQ ID NO:67. 39-48. (canceled)
 49. The polypeptide of claim1, wherein said first antigen binding fragment (AF1) exhibits a higherthermal stability than an anti-CD3 binding fragment consisting of asequence set forth in SEQ ID NO: 206, as evidenced in an in vitro assayby: a higher melting temperature (T_(m)) of said first antigen bindingfragment relative to that of said anti-CD3 binding fragment; or uponincorporating said first antigen binding fragment into a test bispecificantigen binding construct, a higher T_(m) of said test bispecificantigen binding construct relative to that of a control bispecificantigen binding construct, wherein said test bispecific antigen bindingconstruct comprises said first antigen binding fragment and a referenceantigen binding fragment that binds to an antigen other than CD3; andwherein said control bispecific antigen binding construct consists ofsaid anti-CD3 binding fragment consisting of said sequence of SEQ IDNO:206 and said reference antigen binding fragment, optionally whereinsaid Tm of said first antigen binding fragment is at least 2° C.greater, or at least 3° C. greater, or at least 4° C. greater, or atleast 5° C. greater than said Tm of said anti-CD3 binding fragmentconsisting of said sequence of SEQ ID NO:206.
 50. (canceled)
 51. Thepolypeptide of claim 1, wherein: a. said first antigen binding fragment(AF1) comprises a heavy chain variable region (VH_(I)), wherein saidVH_(I) comprises an amino acid sequence having at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or being identicalto an amino acid sequence of SEQ ID NO:102 or 105, b. said first antigenbinding fragment (AF1) comprises a light chain variable region (VL_(I)),wherein said VL_(I) comprises an amino acid sequence having at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or isidentical to an amino acid sequence of any one of SEQ ID NOS: 101, 103,104, 106, or 107: optionally wherein: i. said VHI and said VLI is linkedby a linker comprising an amino acid sequence having at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to asequence set forth in Table A; c. said first antigen binding fragment(AF1) comprises an amino acid sequence having at least 95%, 96%, 97%,98%, 99% sequence identity or being identical to an amino acid sequenceof any one of SEQ ID NOS: 201-205; d. said first antigen bindingfragment (AF1) specifically binds human or cynomolgus monkey (cyno) CD3,optionally wherein said first antigen binding fragment (AF1)specifically binds human CD3, wherein said first antigen bindingfragment (AF1) binds a CD3 complex subunit identified herein by CD3epsilon, CD13 delta, CD3 gamma, or CD3 zeta unit of CD3; e. said firstantigen binding fragment (AF1) binds a CD3 epsilon fragment of CD3; f.said first antigen binding fragment (AF1) exhibits an isoelectric point(pI) that is less than or equal to 6.6: optionally wherein said firstantigen binding fragment (AF1) exhibits an isoelectric point (pI) thatis between 6.0 and 6.6, inclusive; g. said first antigen bindingfragment (AF1) exhibits an isoelectric point (pI) that is at least 0.1,0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 pH units lower than thepI of a reference antigen binding fragment consisting of a sequenceshown in SEQ ID NO: 206; h. said first antigen binding fragment (AF1)specifically binds human or cyno CD3 with a dissociation constant (Kd)constant between about between about 10 nM and about 400 nM, asdetermined in an in vitro antigen-binding assay comprising a human orcyno CD3 antigen; i. said first antigen binding fragment (AF1)specifically binds human or cyno CD3 with a dissociation constant (Kd)of less than about 10 nM, or less than about 50 nM, or less than about100 nM, or less than about 150 nM, or less than about 200 nM, or lessthan about 250 nM, or less than about 300 nM, or less than about 350 nM,or less than about 400 nM, as determined in an in vitro antigen-bindingassay; and/or j. said first antigen binding fragment (AF1) exhibits abinding affinity to CD3 that is at least 2-fold, 3-fold, 4-fold, 5-fold,6-fold, 7-fold, 8-fold, 9-fold, or at least 10-fold weaker relative tothat of an antigen binding fragment consisting of an amino acid sequenceof SEQ ID NO:206, as determined by the respective dissociation constants(Kd) in an in vitro antigen-binding assay. 52-64. (canceled)
 65. Thepolypeptide of claim 1, wherein: a. said first antigen binding fragment(AF1) is: i. a chimeric or a humanized antigen binding fragment; and/orii. is an Fv, Fab, Fab′, Fab′-SH, linear antibody, or single-chainvariable fragment (scFv), b. said second antigen binding fragment (AF2)is an Fv, Fab, Fab′, Fab′-SH, linear antibody, a single domain antibody,or single-chain variable fragment (scFv); c. said first and secondantigen binding fragments are configured as an (Fab′)2 or a single chaindiabody; d. said second antigen binding fragment (AF2) comprises: aheavy chain variable region (VH_(II)) comprising an amino acid sequenceidentified herein by SEQ ID NOS: 778-783, and a light chain variableregion (VL_(II)) comprising an amino acid sequence identified herein bySEQ ID NOS: 878-883; and/or e. said VH_(II) and said VL_(II) is linkedby a linker comprising an amino acid sequence having at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to asequence set forth in Table A. 66-70. (canceled)
 71. The polypeptide ofclaim 1, wherein said first and second antigen binding fragments arefused together by a peptide linker, optionally wherein a. said peptidelinker comprises 2 or 3 types of amino acids are glycine, serine, orproline; and/or b. said peptide linker comprises an amino acid sequencehaving at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%sequence identity to a sequence set forth in Table B.
 72. (canceled) 73.(canceled)
 74. The polypeptide of claim 1, wherein: said XTEN is a firstextended recombinant polypeptide (XTEN1); said plurality ofnon-overlapping sequence motifs, from which said XTEN1 is formed, is afirst plurality of non-overlapping sequence motifs; said BAR is a firstbarcode fragment (BAR1); and said RS is a first release segment (RS1);and the polypeptide further comprising: (d) a second extendedrecombinant polypeptide (XTEN2), comprising: a second barcode fragment(BAR2) releasable from said polypeptide upon digestion by said protease;and (e) a second release segment (RS2) positioned between said secondXTEN (XTEN2) and said bispecific antibody construct (BsAb), wherein saidXTEN2 is characterized in that: (i) it comprises at least 100, or atleast 150 amino acids; (ii) at least 90% of its amino acid residues areglycine (G), alanine (A), serine (S), threonine (T), glutamate (E) orproline (P); and (iii) it comprises at least 4 different types of aminoacids are G, A, S, T, E, or P; wherein said second barcode fragment(BAR2) differs in sequence and molecular weight from all other peptidesfragments that are releasable from said polypeptide upon completedigestion of said polypeptide by said protease; and wherein said secondbarcode fragment (BAR2) does not include said N-terminal amino acid orsaid C-terminal amino acid of said polypeptide, optionally wherein a.said XTEN1 is positioned N-terminal of said bispecific antibodyconstruct and said XTEN2 is positioned C-terminal of said bispecificantibody construct; b. said XTEN1 is positioned C-terminal of saidbispecific antibody construct and said XTEN2 is positioned N-terminal ofsaid bispecific antibody construct.
 75. (canceled)
 76. (canceled) 77.The polypeptide of claim 74, wherein: (iv) said XTEN2 is formed from asecond plurality of non-overlapping sequence motifs that are each from 9to 14 amino acids in length, wherein said second plurality ofnon-overlapping sequence motifs comprise: (1) a second set ofnon-overlapping sequence motifs, wherein each non-overlapping sequencemotif of said second set of non-overlapping sequence motifs is repeatedat least two times in said second XTEN; and (2) a non-overlappingsequence motif that occurs only once within said second XTEN; andwherein said second barcode fragment (BAR2) includes at least part ofsaid non-overlapping sequence motif that occurs only once within saidsecond XTEN; optionally wherein i. said second set of non-overlappingsequence motifs are each independently identified herein by SEQ ID NOS:179-200 and 1715-1722; or ii. said second set of non-overlappingsequence motfis are each independently identified herein by SEQ ID Nos:186-189.
 78. (canceled)
 79. (canceled)
 80. The polypeptide of claim 77,wherein: a. said second set of non-overlapping sequence motifs compriseat least two, at least three, or all four of the sequence motifs SEQ IDNOS: 186-189; b. said XTEN2 comprises a length of from 100 to 3,000,from 150 to 3,000, from 100 to 1,000, or from 150 to 1,000 amino acidresidues; c. said XTEN2 comprises a length of at least 200, at least250, at least 300, at least 350, at least 400, at least 450, or at least500 amino acid residues; and/or d. said XTEN2 has at least 90%, at least92%, at least 95%, at least 98%, at least 99% or 100% sequence identityto a sequence set forth in Table 3a. 81-84. (canceled)
 85. Thepolypeptide of claim 74, wherein: a. said second barcode fragment (BAR2)does not include a glutamic acid that is immediately adjacent to anotherglutamic acid, if present, in said XTEN2; b. said second barcodefragment (BAR2) has a glutamic acid at its C-terminus; c. said secondbarcode fragment (BAR2) has an N-terminal amino acid that is immediatelypreceded by a glutamic acid residue; d. second barcode fragment (BAR2)is positioned a distance from either said N-terminus of said polypeptideor said C-terminus of said polypeptide, wherein said distance is from 10to 150 amino acids, or from 10 to 125 amino acids in length; e. saidsecond barcode fragment (BAR2) is characterized in that: i. it does notinclude a glutamic acid that is immediately adjacent to another glutamicacid, if present, in said XTEN2 ii. it has a glutamic acid at itsC-terminus; iii. it has an N-terminal amino acid that is immediatelypreceded by a glutamic acid residue; and iv. it is positioned a distancefrom either said N-terminus of said polypeptide or said C-terminus ofsaid polypeptide, wherein said distance is from 10 to 150 amino acids,or from 10 to 125 amino acids in length; f. said glutamic acid residuethat precedes said N-terminal amino acid of said BAR2 is not immediatelyadjacent to another glutamic acid residue; g. said second barcodefragment (BAR2) does not include a second glutamic acid residue at aposition other than the C-terminus of said second barcode fragment(BAR2) unless said second glutamic acid is immediately followed by aproline; h. said XTEN2 is positioned N-terminal of said bispecificantibody construct (BsAb), and wherein said second barcode fragment(BAR2) is positioned within 200, within 150, within 100, or within 50amino acids of said N-terminus of said polypeptide; i. said XTEN2 ispositioned N-terminal of said bispecific antibody construct (BsAb), andwherein said second barcode fragment (BAR2) is positioned at a locationthat is between 10 and 200, between 30 and 200, between 40 and 150, orbetween 50 and 100 amino acids from said N-terminus of said protein; j.said XTEN2 is positioned C-terminal of said bispecific antibodyconstruct (BsAb), and wherein said second barcode fragment (BAR2) ispositioned within 200, within 150, within 100, or within 50 amino acidsof said C-terminus of said polypeptide; k. said XTEN2 is positionedC-terminal of said bispecific antibody construct (BsAb), and said secondbarcode fragment (BAR2) is positioned at a location that is between 10and 200, between 30 and 200, between 40 and 150, or between 50 and 100amino acids from said C-terminus of said protein; l. said second barcodefragment (BAR2) is at least 4 amino acids in length, optionally whereinsaid barcode fragment (BAR2) is between 4 and 20, between 5 and 15,between 6 and 12, or between 7 and 10 amino acids in length; m. saidsecond barcode fragment (BAR2) comprises an amino acid sequence setforth in Table 2; n. said XTEN2 has a length defined by a proximal endand a distal end, wherein (1) said proximal end of said XTEN2 ispositioned, relative to said distal end, closer to said bispecificantibody construct (BsAb), and wherein (2) said second barcode fragment(BAR2) is positioned within a region of said XTEN2 that extends, asmeasured from said distal end of said XTEN2, between 5% and 50%, between7% and 40%, or between 10% and 30% of said length of said XTEN2; and/or;o. said XTEN2 further comprises additional one or more barcodefragments, wherein said additional one or more barcode fragments of saidXTEN2 each differ in sequence and molecular weight from all otherpeptides fragments that are releasable from said polypeptide uponcomplete digestion of said polypeptide by said protease. 86-100.(canceled)
 101. The polypeptide of claim 74, wherein: a. said firstrelease segment (RS1) and said second release segment (RS2) areidentical in sequence; b. said first release segment (RS1) and saidsecond release segment (RS2) are not identical in sequence; c. saidsecond release segment (RS) comprises an amino acid sequence having atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequenceidentity to a sequence identified herein by SEQ ID NOS: 7001-7626;and/or d. said first releast segment (RS1) and said second releasesegment (RS2) are each a substrate for cleavage by multiple proteases atone, two, or three cleavage sites within each release segment sequence.102-104. (canceled)
 105. The polypeptide of claim 74 expressed as afusion protein, wherein: a. said fusion protein, in an uncleaved state,has a structural arrangement from N-terminus to C-terminus identifiedherein by XTEN1-RS1-AF1-AF2-RS2-XTEN2, XTEN1-RS1-AF2-AF1-RS2-XTEN2,XTEN2-RS2-AF1-AF2-RS1-XTEN1, XTEN2-RS2-AF2-AF1-RS1-XTEN1,XTEN1-RS1-diabody-RS2-XTEN2, or XTEN2-RS2-diabody-RS1-XTEN1, whereinsaid diabody comprises a light chain variable region (VL_(I)) of saidAF1, a heavy chain variable region (VH_(I)) of said AF1, a light chainvariable region (VL_(II)) of said AF2, and a heavy chain variable region(VH_(II)) of said AF2; b. said spacer of said first release segment(RS1) is a first spacer, and wherein said second release segment (RS2)is fused to said bispecific antibody construct (BsAb) by a secondspacer, optionally wherein i. said second spacer comprises at least 4types of amino acids are glycine (G), alanine (A), serine (S), threonine(T), glutamate (E) or proline (P); or ii. said second spacer comprisesan amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% sequence identity to a sequence set forth inTable C. 106-108. (canceled)
 109. The polypeptide of claim 74, wherein:(a) said XTEN1 comprises an amino acid sequence having at least about90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to a sequence set forth in Table 3a; (b) said BsAb comprises:(I) said AF1 comprising light chain complementarity-determining regions1 (CDR-L1), 2 (CDR-L2), and 3 (CDR-L3) and heavy chaincomplementarity-determining regions 1 (CDR-H1), 2 (CDR-H2), and 3(CDR-H3), wherein said CDR-H1, said CDR-H2, and said CDR-H3 compriseamino acid sequences of SEQ ID NOS: 8, 9, and 10, respectively; and (II)said AF2 comprising a light chain variable region (VL_(II)) identifiedherein by SEQ ID NOS: 778-783 and a heavy chain variable region(VH_(II)) identified herein by SEQ ID NOS: 878-883; (c) said RS1comprises an amino acid sequence having at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequenceidentified herein by SEQ ID NOS: 7001-7626; (d) said XTEN2 comprises anamino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% sequence identity to a sequence set forth inTable 3a; and (e) said RS2 comprises an amino acid sequence having atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to a sequence identified herein by SEQ ID NOS: 7001-7626,wherein said polypeptide has a structural arrangement from N-terminus toC-terminus identified herein by: XTEN1-RS1-AF2-AF1-RS2-XTEN2,XTEN1-RS1-AF1-AF2-RS2-XTEN2, XTEN2-RS2-AF2-AF1-RS1-XTEN1, orXTEN2-RS2-AF1-AF2-RS1-XTEN1.
 110. The polypeptide of claim 1, whereinthe polypeptide: a. has a terminal half-life that is at least two-foldlonger compared to the bispecific antibody construct not linked to anyXTEN; b. is less immunogenic compared to the bispecific antibodyconstruct not linked to any XTEN, as ascertained by measuring productionof IgG antibodies that selectively bind to said bispecific antibodyconstruct after administration of comparable doses to a subject; c.exhibits an apparent molecular weight factor under physiologicalconditions that is greater than about 3, greater than about 4, greaterthan about 5, or greater than about 6; and/or d. comprises an amino acidsequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or 100% sequence identity to a sequence set forth in Table D.111-113. (canceled)
 114. A pharmaceutical composition comprising apolypeptide having an N-terminal amino acid and a C-terminal amino acid,the polypeptide comprising: a. an extended recombinant polypeptide(XTEN), comprising: a barcode fragment (BAR) releasable from saidpolypeptide upon digestion by a protease; b. a bispecific antibodyconstruct (BsAb), comprising: a first antigen binding fragment (AF1)that specifically binds to cluster of differentiation 3 T cell receptor(CD3), which AF1 comprises light chain complementarity-determiningregions 1 (CDR-L1), 2 (CDR-L2), and 3 (CDR-L3) and heavy chaincomplementarity-determining regions 1 (CDR-H1), 2 (CDR-H2), and 3(CDR-H3), wherein said CDR-H3 comprises an amino acid sequence of SEQ IDNO:10; and a second antigen binding fragment (AF2) that specificallybinds to human epidermal growth factor receptor 2 (HER2); and c. arelease segment (RS) positioned between said XTEN and said bispecificantibody construct, wherein said XTEN is characterized in that: (i) itcomprises at least 100, or at least 150 amino acids; (ii) at least 90%of its amino acid residues are identified herein by glycine (G), alanine(A), serine (S), threonine (T), glutamate (E) or proline (P); (iii) itcomprises at least 4 different types of amino acids identified herein byG, A, S, T, E, or P; and (iv) said XTEN is formed from a plurality ofnon-overlapping sequence motifs that are each from 9 to 14 amino acidsin length, wherein said plurality of non-overlapping sequence motifscomprise: (1) a set of non-overlapping sequence motifs, wherein eachnon-overlapping sequence motif of said set of non-overlapping sequencemotifs is repeated at least two times in said XTEN; and (2) anon-overlapping sequence motif that occurs only once within said XTEN;wherein said barcode fragment (BAR) includes at least part of saidnon-overlapping sequence motif that occurs only once within said XTEN;wherein said barcode fragment (BAR) differs in sequence and molecularweight from all other peptides fragments that are releasable from saidpolypeptide upon complete digestion of said polypeptide by saidprotease; and wherein said barcode fragment (BAR) does not include saidN-terminal amino acid or said C-terminal amino acid of said polypeptide;and one or more pharmaceutically suitable excipients.
 115. Thepharmaceutical composition of claim 114, wherein said pharmaceuticalcomposition: a. is formulated for intradermal, subcutaneous, oral,intravenous, intra-arterial, intraabdominal, intraperitoneal,intrathecal, or intramuscular administration; b. is in a liquid form orfrozen; c. is in a pre-filled syringe for a single injection, optionallywherein said pharmaceutical composition is formulated as a lyophilizedpowder to be reconstituted prior to administration; and/or d. is in apharmaceutical combination comprising at least one additionaltherapeutic agent selected form the group consisting of an antibody, anantibody fragment, an antibody conjugate, a cytotoxic agent, a toxin, aradionuclide, an immunomodulator, a photoactive therapeutic agent, aradiosensitizing agent, a hormone, an anti-angiogenesis agent, andcombinations thereof; optionally wherein: i. said additional therapeuticagent is a PD-1/PD-L1(2) inhibitor; ii. the PD-1/PD-L1(2) inhibitor isan anti-PD-1 antibody or an anti-PD-L1 antibody or an anti-PD-L2antibody; iii. the PD-1/PD-L1(2) is an anti-PD-1 antibody selected fromthe group comprising nivolumab (Opdivo, BMS-936558, MDX1106),pembrolizumab (Keytruda, MK-3475, lambrolizumab), pidilizumab (CT-011),PDR-001, JS001, STI-A1110, AMP-224 and AMP-514 (MEDI0680); and/or iv.the PD-1/PD-L1(2) inhibitor is an anti-PD-L1 antibody selected from thegroup comprising atezolizumab (Tecentriq, MPDL3280A), durvalumab(MEDI4736), avelumab (MSB0010718C), BMS-936559 (MDX11105) and LY3300054.116-123. (canceled)
 124. The pharmaceutical composition of claim 114,wherein: a. the PD-1/PD-L1(2) inhibitor is an anti-PD-L2 antibody; b.the components are administered in separate dosage forms simultaneouslyor sequentially for use in the treatment of the same disease; c. are foruse as medicament for treating hyper-proliferative disorder, optionallywherein the hyper-proliferative disorders are selected from the groupconsisting of cancers of the breast, respiratory tract, brain,reproductive organs, digestive tract, urinary tract, eye, liver, skin,head and neck, thyroid, parathyroid and their distant metastases.125-130. (canceled)
 131. A method of treating a disease in a subject,comprising administering to said subject in need thereof one or moretherapeutically effective doses of a pharmaceutical compositioncomprising a polypeptide comprising having an N-terminal amino acid anda C-terminal amino acid, the polypeptide comprising: a. an extendedrecombinant polypeptide (XTEN), comprising: a barcode fragment (BAR)releasable from said polypeptide upon digestion by a protease; b. abispecific antibody construct (BsAb), comprising: a first antigenbinding fragment (AF1) that specifically binds to cluster ofdifferentiation 3 T cell receptor (CD3), which AF1 comprises light chaincomplementarity-determining regions 1 (CDR-L1), 2 (CDR-L2), and 3(CDR-L3) and heavy chain complementarity-determining regions 1 (CDR-H1),2 (CDR-H2), and 3 (CDR-H3), wherein said CDR-H3 comprises an amino acidsequence of SEQ ID NO:10; and a second antigen binding fragment (AF2)that specifically binds to human epidermal growth factor receptor 2(HER2); and c. a release segment (RS) positioned between said XTEN andsaid bispecific antibody construct, wherein said XTEN is characterizedin that: (i) it comprises at least 100, or at least 150 amino acids;(ii) at least 90% of its amino acid residues are identified herein byglycine (G), alanine (A), serine (S), threonine (T), glutamate (E) orproline (P); (iii) it comprises at least 4 different types of aminoacids identified herein by G, A, S, T, E, or P; and (iv) said XTEN isformed from a plurality of non-overlapping sequence motifs that are eachfrom 9 to 14 amino acids in length, wherein said plurality ofnon-overlapping sequence motifs comprise: (1) a set of non-overlappingsequence motifs, wherein each non-overlapping sequence motif of said setof non-overlapping sequence motifs is repeated at least two times insaid XTEN; and (2) a non-overlapping sequence motif that occurs onlyonce within said XTEN; wherein said barcode fragment (BAR) includes atleast part of said non-overlapping sequence motif that occurs only oncewithin said XTEN; wherein said barcode fragment (BAR) differs insequence and molecular weight from all other peptides fragments that arereleasable from said polypeptide upon complete digestion of saidpolypeptide by said protease; and wherein said barcode fragment (BAR)does not include said N-terminal amino acid or said C-terminal aminoacid of said polypeptide.
 132. The method of claim 131, wherein: a. saiddisease is cancer; b. said cancer is selected from the group consistingof glioblastoma, melanoma, cholangio carcinoma, small cell lung cancer,colorectal cancer, prostate cancer, vaginal cancer, angiosarcoma,non-small cell lung cancer, appendiceal cancer, squamous cell cancer,salivary duct carcinoma, adenoid cystic carcinoma, small intestinecancer, and gallbladder cancer; c. the pharmaceutical composition isadministered to the subject as one or more therapeutically effectivedoses; d. the pharmaceutical composition is administered to the subjectas one or more therapeutically effective doses over an effective dosingperiod; and/or e. the subject is a mouse, rate, monkey, or human.133-136. (canceled)
 137. A nucleic acid comprising (a) a polynucleotidesequence encoding a polypeptide having an N-terminal amino acid and aC-terminal amino acid, the polypeptide comprising: a. an extendedrecombinant polypeptide (XTEN), comprising: a barcode fragment (BAR)releasable from said polypeptide upon digestion by a protease; b. abispecific antibody construct (BsAb), comprising: a first antigenbinding fragment (AF1) that specifically binds to cluster ofdifferentiation 3 T cell receptor (CD3), which AF1 comprises light chaincomplementarity-determining regions 1 (CDR-L1), 2 (CDR-L2), and 3(CDR-L3) and heavy chain complementarity-determining regions 1 (CDR-H1),2 (CDR-H2), and 3 (CDR-H3), wherein said CDR-H3 comprises an amino acidsequence of SEQ ID NO:10; and a second antigen binding fragment (AF2)that specifically binds to human epidermal growth factor receptor 2(HER2); and c. a release segment (RS) positioned between said XTEN andsaid bispecific antibody construct, wherein said XTEN is characterizedin that: (i) it comprises at least 100, or at least 150 amino acids;(ii) at least 90% of its amino acid residues are identified herein byglycine (G), alanine (A), serine (S), threonine (T), glutamate (E) orproline (P); (iii) it comprises at least 4 different types of aminoacids identified herein by G, A, S, T, E, or P; and (iv) said XTEN isformed from a plurality of non-overlapping sequence motifs that are eachfrom 9 to 14 amino acids in length, wherein said plurality ofnon-overlapping sequence motifs comprise: (1) a set of non-overlappingsequence motifs, wherein each non-overlapping sequence motif of said setof non-overlapping sequence motifs is repeated at least two times insaid XTEN; and (2) a non-overlapping sequence motif that occurs onlyonce within said XTEN; wherein said barcode fragment (BAR) includes atleast part of said non-overlapping sequence motif that occurs only oncewithin said XTEN; wherein said barcode fragment (BAR) differs insequence and molecular weight from all other peptides fragments that arereleasable from said polypeptide upon complete digestion of saidpolypeptide by said protease; and wherein said barcode fragment (BAR)does not include said N-terminal amino acid or said C-terminal aminoacid of said polypeptide; or (b) a reverse complement of saidpolynucleotide sequence of (a).
 138. An expression vector comprisingsaid polynucleotide sequence of claim 137 and a recombinant regulatorysequence operably linked to said polynucleotide sequence.
 139. A hostcell, comprising said expression vector of claim 138, optionally whereinthe host cell is a prokaryote, E. coli, or a mammalian cell. 140-142.(canceled)